Antibacterial antisense oligonucleotide and method

ABSTRACT

A method for enhancing, by at least 10 fold, the antibacterial activity of an antisense oligonucleotide composed of morpholino subunits linked by phosphorus-containing intersubunit linkages. The method includes one or both of: conjugating an arginine-rich carrier to a 3′ or 5′ end of the oligonucleotide and modifying the oligonucleotide to contain 20%-50% intersubunit linkages that are positively charged at physiological pH. Also disclosed is an antisense oligonucleotide having enhanced antibacterial activity by virtue of one or both modifications.

This application claims priority to U.S. patent application Ser. No.11/487,009 filed on Jul. 13, 2006, which claims priority to U.S.Provisional Patent Application No. 60/699,280 filed on Jul. 13, 2005,now abandoned, both of which are incorporated herein by reference intheir entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 120178_438C5_SEQUENCE_LISTING_ST25.txt. The textfile is 24.1 KB, was created on Dec. 21, 2015, and is being submittedelectronically via EFS-Web.

FIELD OF THE INVENTION

The present invention relates to peptide-conjugated morpholinooligonucleotide compounds having partially charged backbones and thatare antisense to bacterial genes, and methods for use of such compoundsin inhibiting bacterial growth, e.g., in an infected mammalian subject.

REFERENCES

-   Anderson, K. P., M. C. Fox, et al. (1996). Antimicrob Agents    Chemother 40(9): 2004-11.-   Bramhill, D. (1997). Annu Rev Cell Dev Biol 13: 395-424.-   Donachie, W. D. (1993). Annu Rev Microbiol 47: 199-230.-   Geller, B. L., J. D. Deere, et al. (2003). Antimicrob Agents    Chemother 47(10): 3233-9.-   Geller, B. L. and H. M. Green (1989). J Biol Chem 264(28): 16465-9.-   Gerdes, S. Y., M. D. Scholle, et al. (2003). J Bacteriol 185(19):    5673-84.-   Good, L., S. K. Awasthi, et al. (2001). Nat Biotechnol 19(4): 360-4.-   Hale, C. A. and P. A. de Boer (1999). J Bacteriol 181(1): 167-76.-   Lutkenhaus, J. and S. G. Addinall (1997). Annu Rev Biochem 66:    93-116.-   Pari, G. S., A. K. Field, et al. (1995). Antimicrob Agents Chemother    39(5): 1157-61.-   Summerton, J., D. Stein, et al. (1997). Antisense Nucleic Acid Drug    Dev 7(2): 63-70.-   Summerton, J. and D. Weller (1997). Antisense Nucleic Acid Drug Dev    7(3): 187-95.-   Zhang, Y. and J. E. Cronan, Jr. (1996). J Bacteriol 178(12):    3614-20.

BACKGROUND OF THE INVENTION

Currently, there are several types of antibiotic compounds in useagainst bacterial pathogens, and these compounds act through a varietyof anti-bacterial mechanisms. For example, beta-lactam antibiotics, suchas penicillin and cephalosporin, act to inhibit the final step inpeptidoglycan synthesis. Glycopeptide antibiotics, including vancomycinand teicoplanin, inhibit both transglycosylation and transpeptidation ofmuramyl-pentapeptide, again interfering with peptidoglycan synthesis.Other well-known antibiotics include the quinolones, which inhibitbacterial DNA replication, inhibitors of bacterial RNA polymerase, suchas rifampin, and inhibitors of enzymes in the pathway for production oftetrahydrofolate, including the sulfonamides.

Some classes of antibiotics act at the level of protein synthesis.Notable among these are the aminoglycosides, such as kanamycin andgentamycin. This class of compounds targets the bacterial 30S ribosomesubunit, preventing the association with the 50S subunit to formfunctional ribosomes. Tetracyclines, another important class ofantibiotics, also target the 30S ribosome subunit, acting by preventingalignment of aminoacylated tRNA's with the corresponding mRNA codon.Macrolides and lincosamides, another class of antibiotics, inhibitbacterial synthesis by binding to the 50S ribosome subunit, andinhibiting peptide elongation or preventing ribosome translocation.

Despite impressive successes in controlling or eliminating bacterialinfections by antibiotics, the widespread use of antibiotics both inhuman medicine and as a feed supplement in poultry and livestockproduction has led to drug resistance in many pathogenic bacteria.Antibiotic resistance mechanisms can take a variety of forms. One of themajor mechanisms of resistance to beta lactams, particularly inGram-negative bacteria, is the enzyme beta-lactamase, which renders theantibiotic inactive. Likewise, resistance to aminoglycosides ofteninvolves an enzyme capable of inactivating the antibiotic, in this caseby adding a phosphoryl, adenyl, or acetyl group. Active efflux ofantibiotics is another way that many bacteria develop resistance. Genesencoding efflux proteins, such as the tetA, tetG, tetL, and tetK genesfor tetracycline efflux, have been identified. A bacterial target maydevelop resistance by altering the target of the drug. For example, theso-called penicillin binding proteins (PBPs) in many beta-lactamresistant bacteria are altered to inhibit the critical antibioticbinding to the target protein. Resistance to tetracycline may involve,in addition to enhanced efflux, the appearance of cytoplasmic proteinscapable of competing with ribosomes for binding to the antibiotic. Forthose antibiotics that act by inhibiting a bacterial enzyme, such as forsulfonamides, point mutations in the target enzyme may conferresistance.

The appearance of antibiotic resistance in many pathogenic bacteria—inmany cases involving multi-drug resistance—has raised the specter of apre-antibiotic era in which many bacterial pathogens are simplyuntreatable by medical intervention. There are two main factors thatcould contribute to this scenario. The first is the rapid spread ofresistance and multi-resistance genes across bacterial strains, species,and genera by conjugative elements, the most important of which areself-transmissible plasmids. The second factor is a lack of currentresearch efforts to find new types of antibiotics, due in part to theperceived investment in time and money needed to find new antibioticagents and bring them through clinical trials, a process that mayrequire a 20-year research effort in some cases.

In addressing the second of these factors, some drug-discoveryapproaches that may accelerate the search for new antibiotics have beenproposed. For example, efforts to screen for and identify new antibioticcompounds by high-throughput screening have been reported, but to dateno important lead compounds have been discovered by this route.

Several approaches that involve antisense agents designed to block theexpression of bacterial resistance genes or to target cellular RNAtargets, such as the rRNA in the 30S ribosomal subunit (see, forexample, co-owned U.S. Pat. No. 6,677,153, which is incorporated byreference herein), or by targeting the mRNA of bacterial proteins thatare critical in bacterial replication, such as acyl carrier protein(acpP), gyrase A subunit (gyrA), or the cell division protein ftsZ (seeco-owned U.S. patent applications 20070021362 and 20070049542, both ofwhich are incorporated by reference herein). Although these approacheshave been shown to be successful in blocking bacterial replication, theyhave been limited in commercial applications by the concentrations ofantisense compounds required for efficacy in treating bacterialinfections in a mammalian host.

There is thus a continuing need for new antibiotics that (i) are notsubject to the principal types of antibiotic resistance currentlyhampering antibiotic treatment of bacteria, (ii) can be developedrapidly and with some reasonable degree of predictability as totarget-bacteria specificity, (iii) are effective at low doses, and (iv)show relatively few side effects.

SUMMARY OF THE INVENTION

The invention includes, in one aspect, a method for enhancing theantibacterial activity of an antisense oligonucleotide composed ofmorpholino subunits linked by phosphorus-containing intersubunitlinkages joining a morpholino nitrogen of one subunit to a 5′ exocycliccarbon of an adjacent subunit, where the oligonucleotide containsbetween 10-20 bases and a targeting sequence of at least 10 contiguousbases complementary to a bacterial RNA target, and where binding of theoligonucleotide to the RNA target region is effective to inhibit growthof an infectious bacterium in a mammalian host. The method includes oneor both of the steps of:

(a) conjugating to the oligonucleotide, a carrier peptide (i) containing6-16, preferably 8-14 amino acids composed of the subsequences selectedfrom the group represented by XXY, XY, XZZ and XZ, where (a) each Xsubunit independently represents arginine or an arginine analog, saidanalog being a cationic α-amino acid comprising a side chain of thestructure R1N═C(NH2)R2, where R1 is H or R; R2 is R, NH2, NHR, or NR2,where R is lower alkyl or lower alkenyl and may further include oxygenor nitrogen; R1 and R2 may together form a ring; and the side chain islinked to said amino acid via R1 or R2; (b) each Y subunit independentlyrepresents a neutral linear amino acid —C(O)—(CHR)m-NH—, where (i) m is1 to 7 and each R is independently H or methyl, and (c) Z is an α-aminoacid having a neutral side chain selected from a substituted orunsubstituted aralkyl, and (ii) coupled to the oligonucleotide at thepeptide's C terminus, and

(b) modifying the oligonucleotide to contain 10%-80%, preferably 20%-50%intersubunit cationic linkages that are positively charged atphysiological pH.

The carrier peptide in step (a) may be represented by the sequence(RY′R)_(n), or (RY′)_(n), where R is arginine and Y′ is a linearalkanoic acid having 2-7 carbon atoms in its backbone chain, such as thepeptides (RAhxR)₄, where Ahx is 6-amino hexanoic acid; and (RAhx)₆,where Ahx is 6-amino hexanoic acid.

The carrier peptide in step (a) may be linked at its C-terminus to the5′ end of the oligonucleotide through a one- or two-amino acid linker.Exemplary linkers include AhxβAla, where Ahx is 6-aminohexanoic acid andβAla is β-alanine. Carrier peptide length, e.g., 8-14, is consideredindependent of the presence of one or more amino-acid linkers.

The enhancement in activity produced by step (a) alone or step (b) alonemay be effective to enhance the anti-bacterial activity of an unchargedoligonucleotide, as measured by inhibition in bacterial growth in vitroover an eight-hour period, by a factor of at least 10 relative to themeasured inhibition of the uncharged oligonucleotide in the absence ofthe carrier peptide. The enhancement in activity produced by step (a)and step (b) together may be effective to enhance the anti-bacterialactivity of the peptide-conjugated oligonucleotide, as measured byinhibition in bacterial growth in vitro over an eight-hour period, by afactor of at least 10² relative to the measured inhibition of theuncharged oligonucleotide in the absence of the carrier peptide.

The morpholino subunits in the oligonucleotide may be joined byphosphorodiamidate linkages, including both uncharged and cationiclinkages, in accordance with the structure:

where

Z is S or O,

X═NR¹R² or OR⁶,

Y═O or NR⁷,

and each said linkage is selected from:

(a) uncharged linkage (a), where each of R¹, R², R⁶ and R⁷ isindependently selected from hydrogen and lower alkyl;

(b1) cationic linkage (b1), where X═NR¹R² and Y═O, and NR¹R² representsan optionally substituted piperazino group, such thatR¹R²═—CHRCHRN(R³)(R⁴)CHRCHR—, where

each R is independently H or CH₃,

R⁴ is H, CH₃ or an electron pair, and

R³ is selected from H, lower alkyl, C(═NH)NH₂, Z-L-NHC(═NH)NH₂, and[C(O)CHR′NH]_(m)H, where Z is carbonyl (C(O)) or a direct bond, L is anoptional linker up to 18 atoms in length having bonds selected fromalkyl, alkoxy, and alkylamino, R′ is a side chain of a naturallyoccurring amino acid or a one- or two-carbon homolog thereof, and m is 1to 6;

(b2) cationic linkage (b2), where X═NR¹R² and Y═O, R¹═H or CH₃, andR²=LNR³R⁴R⁵, where L, R³, and R⁴ are as defined above, and R⁵ is H,lower alkyl, or lower (alkoxy)alkyl; and

(b3) cationic linkage (b3), where Y═NR⁷ and X═OR⁶, and R⁷=LNR³R⁴R⁵,where L, R³, R⁴ and R⁵ are as defined above, and R⁶ is H or lower alkyl;and at least one said linkage is selected from cationic linkages (b1),(b2), and (b3);

In various embodiments of the backbone linkages: (i) each of R¹ and R²,in linkages of type (a), is methyl; (ii) at least one linkage is of type(b1), and each R is H, R⁴ is H, CH₃, or an electron pair, and R³ isselected from H, CH₃, C(═NH)NH₂, and C(O)-L-NHC(═NH)NH₂; (iii) at leastone linkage is of type (b1), and each R is H, R⁴ is an electron pair,and R³ is selected from C(═NH)NH₂ and C(O)-L-NHC(═NH)NH₂; (iv) at leastone linkage is of type (b1), and each R is H, R⁴ is an electron pair,and R³ is selected from C(═NH)NH₂ and C(O)-L-NHC(═NH)NH₂, and moreparticularly, R³ is C(O)-L-NHC(═NH)NH2, and L is a hydrocarbon havingthe structure —(CH₂)_(n)—, where n is 1 to 12; (v) at least one linkageis of type (b1), and each R is H, and each of R³ and R⁴ is independentlyH or CH₃.

Considering the oligonucleotide to have approximately equal-length 5′,3′ and center regions, and the percentage of cationic linkages in step(b) said center region may be greater than about 70%.

Where the antisense oligonucleotide is intended for use in treating agram-negative bacterial infection, the targeting sequence of theoligonucleotide may be complementary to a target sequence containing orwithin 20 bases, in a downstream direction, of the translational startcodon of a bacterial mRNA that encodes acyl carrier protein (acpP). Forexample, the targeting sequence may be complementary to at least tencontiguous bases in a sequence selected from the group consisting of SEQID NOS: 2, 5, 8, 11, 14, 17, 20, 23, 28, 31, 34, 36, 39, 42, 45, 48, 51,54, 57 and 60.

Where the antisense oligonucleotide is intended for use treating agram-negative bacterial infection, wherein the targeting sequence iscomplementary to a target sequence containing or within 20 bases, in adownstream direction, of the translational start codon of a bacterialmRNA that encodes gyrase A subunit (gyrA). For example, the targetingsequence may be complementary to at least ten contiguous bases in asequence selected from the group consisting of SEQ ID NOS: 3, 6, 9, 12,15, 18, 24, 29, 32, 35, 37, 40, 43, 46, 49, 52, 55, 58 and 61.

Where the antisense oligonucleotide is intended for targeting abacterial mRNA encoding a bacterial ftsZ protein, the compound targetingsequence may be complementary to at least ten contiguous bases in asequence selected from the group consisting of SEQ ID NOS: 1, 4, 7, 10,13, 16, 19, 22, 27, 30, 33, 38, 41, 44, 47, 50, 53, 56 and 59.

Other bacterial RNA targets may include the AUG start site regions,e.g., the AUG start site itself and up to 15 bases downstream thereof,of a variety of other essential bacterial proteins, and the 16S or 23SrRNA of the 30S bacterial ribosomal subunit.

In another aspect, the invention includes an improved antisenseoligonucleotide useful for treating a bacterial infection, where theoligonucleotide is composed of morpholino subunits linked byphosphorus-containing intersubunit linkages joining a morpholinonitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit,where the oligonucleotide contains between 10-20 bases and a targetingsequence of at least 10 contiguous bases complementary to a bacterialRNA target, and where binding of the oligonucleotide to the RNA targetregion is effective to inhibit growth of the infectious bacterium. Theimprovement includes one or both of the modifications (a) and (b):

(a) conjugated to the oligonucleotide, a carrier peptide that is (i)represented by the sequence selected from the group consisting of(XYX)_(n), (XY)_(n) and (XZZ)_(n), where (a) each X subunitindependently represents arginine or an arginine analog, said analogbeing a cationic α-amino acid comprising a side chain of the structureR1N═C(NH2)R2, where R1 is H or R; R2 is R, NH2, NHR, or NR2, where R islower alkyl or lower alkenyl and may further include oxygen or nitrogen;R1 and R2 may together form a ring; and the side chain is linked to saidamino acid via R1 or R2; (b) each Y subunit independently represents aneutral linear amino acid —C(O)—(CHR)m-NH—, where (i) m is 1 to 7 andeach R is independently H or methyl, or (ii) m is 1 and R is a neutralside chain selected from substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, and aralkyl, wherein said neutral side chain, whenselected from substituted alkyl, alkenyl, and alkynyl, includes at mostone heteroatom for every four carbon atoms; and (c) each Z subunitindependently represents an amino acid selected from alanine,asparagine, cysteine, glutamine, glycine, histidine, lysine, methionine,serine, and threonine, and n is selected to yield a total of 8-14 aminoacids in the peptide sequence, and (ii) coupled to the oligonucleotideat the peptide's C terminus, and

(b) the presence in the oligonucleotide of 10%-80%, preferably 20%-50%intersubunit cationic linkages that are positively charged atphysiological pH.

Also discloses is a method for treating a bacterial infection using theimproved antisense oligonucleotide.

These and other objects and features of the claimed subject matter willbecome more fully apparent when the following detailed description isread in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-D show the repeating subunit segment of exemplary morpholinooligonucleotides, designated A through D, constructed using subunitshaving 5-atom (A), six-atom (B) and seven-atom (C-D) linking groupssuitable for forming polymers.

FIG. 2A shows representative morpholino subunits 1a-e with protectedrecognition moieties P_(i) of A, C, G, T, and I.

FIG. 2B shows synthetic schemes for preparation of the subunits of FIG.2A from the corresponding ribonucleosides.

FIG. 2C illustrates the preparation of activated, protected subunits forpreparation of linkage type (a) (uncharged) and linkage types (b1) and(b2) (charged) as designated herein.

FIG. 2D is a schematic of a synthetic pathway that can be used to makemorpholino subunits containing the (1-piperazino) phosphinylideneoxy(“Pip”) linkage.

FIGS. 2E and 2F illustrate the preparation of activated, protectedsubunits for preparation of linkages of type (b3) (charged) asdesignated herein.

FIG. 2G illustrates the preparation of subunits that can be used toprepare linkages analogous to type (b3) (charged) but based onnon-phosphorus-containing linkages, specifically sulfonamide linkages.

FIG. 2H illustrates preparation of a disulfide anchor, for use inmodification of a synthesis resin used for stepwise preparation of amorpholino oligomer, allowing facile release of the oligomer bytreatment with a thiol.

FIG. 2I illustrates the introduction a triethylene glycol containingmoiety (“Tail”) which increases aqueous solubility of syntheticantisense oligomers.

FIG. 2J illustrates the preparation of resins useful for the solid phasesynthesis of morpholino oligomers.

FIG. 2K illustrates the preparation of N2,O6-protected morpholino GSubunit for large scale oligomer synthesis

FIG. 2L illustrates the introduction of guanidinium groups by directguanylation of amines on the morpholino oligomer.

FIG. 2M illustrates the introduction of guanidinium groups intomorpholino oligomers by incorporation of amino acids and guanidinoacids.

FIG. 2N illustrates the introduction of guanidinium groups intomorpholino oligomers by incorporation of guanidino acids at bothbackbone and terminal positions.

FIG. 2O illustrates the introduction of peptides into the backbonemorpholino oligomers.

FIG. 2P illustrates the introduction of a transport peptides at the3′-terminus of morpholino oligomers having charged groups of linkagetype b1 in the backbone.

FIG. 2Q illustrates the introduction of a transport peptides at the3′-terminus of morpholino oligomers having GuX linkages in the backbone.

FIG. 2R illustrates the reductive alkylation of amines of morpholinooligomers.

FIGS. 3A and 3B show the effect of AcpP antisense length on growth of E.coli AS19. Cultures of E. coli AS19 were grown (37° C.) with variouslengths (6 to 20 bases) of overlapping PMO (20 μM) targeted to theregion around the start codon of the E. coli acpP (Table 2, SEQ IDNO:2). Optical density (OD) was monitored over time (FIG. 3A) and opensquares indicate culture with 11-base PMO 169 (SEQ ID NO:66) and viablecells (CFU/ml) measured after 8 hours (FIG. 3B).

FIG. 4 shows the effect of antisense length on AcpP-luciferaseexpression in cell-free translation reactions. PMOs of various lengthsand targeted around the start codon of acpP (Table 2) were addedindividually (100 nM) to bacterial cell-free translation reactionsprogrammed to make AcpP-luc.

FIG. 5 shows CFU/ml in peritoneal lavages from mice infected withpermeable E. coli strain AS19 and treated with acpP PMO (▪; SEQ IDNO:66), nonsense PMO (▴), or PBS (▾) at 0 hours. At each time indicated,peritoneal lavage was collected and analyzed for bacteria (CFU/ml) from3 mice in each treatment group.

FIG. 6 shows CFU/ml in peritoneal lavages from mice infected E. colistrain SM105 and treated with PMO as described in FIG. 13.

FIGS. 7 to 10 show the growth as measured by optical density (OD₆₀₀) offour strains of E. coli grown for 24 hours in the presence of thepeptide-conjugated PMO (P-PMO) RFF-AcpP11 (SEQ ID NO:79) compared to notreatment and treatment with a mixture of the AcpP11 μMO and the RFFpeptide (SEQ ID NOS:66 and 79, respectively).

FIG. 11 shows the colony-forming units per milliliter (CFU/ml) of fourstrains of E. coli after eight hours incubation in the presence of theRFF-AcpP11 P-PMO compared to no treatment and treatment with a mixtureof the AcpP11 μMO and the RFF peptide.

FIG. 12 shows the antibacterial activity of three P-PMOs as measured byCFU/ml after 8 hours of treatment.

FIG. 13 shows the effect of treatment with a dilution series of RFFpeptide, free RFF peptide mixed with AcpP11 μMO, RFF-AcpP11 P-PMO,ampicillin or no treatment.

FIG. 14 shows the dose response curves for each of two P-PMOs comparedto ampicillin and the associated IC₅₀ values for RFF-AcpP11, RTR-AcpP11(SEQ ID NOS:88 and 89) and ampicillin.

FIGS. 15 and 16 show the effect of RFF-AcpP11 P-PMO on S. typhimuriumand an enteropathogenic strain of E. coli (O127:H6) as measured byCFU/ml after eight hours of treatment compared to no treatment, AcpP11alone, RFF peptide alone, scrambled controls, and a mixture of AcpP11and RFF peptide.

FIGS. 17 and 18 show the effect of RFFR-conjugated P-PMOs on the growthof Burkholderia cenocepacia and Pseudomonas aeruginosa, respectively.

FIGS. 19 A-C show the dose response antibacterial effect of the(RAhxR)₄-AcpP11+P-PMO+ on E. coli infected mice up to 48 hours postinfection as measured by percent survival of mice (FIG. 19A), CFU/mL inmouse blood samples (19B), and change in body temperature (FIG. 19C);

FIG. 20 shows the effect of (RAhxR)₄-AcpP11+P-PMO+ compared toampicillin on wild type E. coli as measured by minimum inhibitoryconcentration (MIC).

DETAILED DESCRIPTION I. Definitions

The terms below, as used herein, have the following meanings, unlessindicated otherwise:

As used herein, the terms “compound”, “agent”, “oligomer” and“oligonucleotide” may be used interchangeably with respect to theantisense oligonucleotides of the claimed subject matter.

As used herein, the terms “antisense oligonucleotide” and“oligonucleotide” or “antisense compound” or “oligonucleotide compound”are used interchangeably and refer to a sequence of subunits, eachhaving a base carried on a backbone subunit composed of morpholinobackbone groups, and where the backbone groups are linked byintersubunit linkages (both charged and uncharged) that allow the basesin the compound to hybridize to a target sequence in an RNA byWatson-Crick base pairing, to form an RNA:oligonucleotide heteroduplexwithin the target sequence. The oligonucleotide may have exact sequencecomplementarity to the target sequence or near complementarity. Suchantisense oligonucleotides are designed to block or inhibit translationof the mRNA containing the target sequence, or to inhibit bacterialprotein synthesis by binding to bacterial 16S or 23S rRNA, and may besaid to be “directed to” a sequence with which it hybridizes. Exemplarystructures for antisense oligonucleotides for use in the claimed subjectmatter in include the morpholino subunit types shown in FIGS. 1A-D.

The term “oligonucleotide” or “antisense oligonucleotide” alsoencompasses an oligonucleotide having one or more additional moietiesconjugated to the oligonucleotide, e.g., at its 3′- or 5′-end, such as apolyethyleneglycol moiety or other hydrophilic polymer, e.g., one having10-100 monomeric subunits, which may be useful in enhancing solubility.

A carrier peptide conjugated to an antisense oligonucleotide, e.g., bycovalent linkage between the peptide's C terminal end and the 5′- or3′-end of the oligonucleotide, is separately named, and typically notincluded within the term “oligonucleotide,” unless understood otherwisefrom the context of the statement. The carrier peptide and covalentlyattached antisense oligonucleotide are also referred to herein as aconjugate or conjugate compound. More generally, a “peptide-conjugatedmorpholino antisense oligonucleotide” is a morpholino antisenseoligonucleotide conjugated at either its 5′ or 3′ termini to anarginine-rich peptide carrier.

By “arginine-rich carrier peptide” is meant that the carrier peptide hasat least 2 arginine residues and preferably 50% or more arginine orarginine-analog residues (Arg residue), each Arg residue or ArgArgresidue pair preferably being separated by one or more uncharged,preferably non-polar amino acid residues. Preferred arginine-richcarrier peptides in the invention contain 8-14 amino acids composed ofthe subsequences selected from the group represented by XXY, XY, XZZ andXZ, where (a) each X subunit independently represents arginine or anarginine analog (“Arg residue”), said analog being a cationic α-aminoacid comprising a side chain of the structure R1N═C(NH2)R2, where R1 isH or R; R2 is R, NH2, NHR, or NR2, where R is lower alkyl or loweralkenyl and may further include oxygen or nitrogen; R1 and R2 maytogether form a ring; and the side chain is linked to said amino acidvia R1 or R2; (b) each Y subunit independently represents a neutrallinear amino acid —C(O)—(CHR)m-NH—, where (i) m is 1 to 7, such asβ-alanine (m=2) and 6-amino hexanoic acid (m=6), and each R isindependently H or methyl, and (c) Z is an α-amino acid having a neutralside chain selected from a substituted or unsubstituted aralkyl, such asphenylalanine, and (ii) coupled to the oligonucleotide at the peptide'sC terminus. Exemplary arginine rich peptides are listed as SEQ IDNOS:79-87.

The carrier peptide may be linked at its C-terminus to one end of theoligonucleotide, e.g., the 5′-end, through a one- or two-amino acidlinker, such as the linker is AhxβAla, where Ahx is 6-aminohexanoic acidand βAla is β-alanine, and where the linker forms part of the carrierpeptide.

As used herein, a “morpholino oligomer” or “morpholino oligonucleotide”refers to an antisense oligonucleotide having a backbone which supportsbases capable of hydrogen bonding to natural polynucleotides, e.g., DNAor RNA, is composed of morpholino subunit structures of the form shownin FIGS. 1A-1D, where (i) the structures are linked together byphosphorous-containing linkages, one to three atoms long, joining themorpholino nitrogen of one subunit to the 5′ exocyclic carbon of anadjacent subunit, and (ii) P_(i) and P_(j) are purine or pyrimidinebase-pairing moieties effective to bind, by base-specific hydrogenbonding, to a base in a polynucleotide. Various subunit and backbonelinkage structures in an oligonucleotide composed of morpholino subunitslinked by phosphorus-containing intersubunit linkages are shown in FIGS.1A-1D, as described further below. Morpholino oligonucleotides of thistype (including antisense oligonucleotides) are detailed, for example,in co-owned U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506,5,166,315, 5,185,444, 5,521,063, and 5,506,337, all of which areexpressly incorporated by reference herein. FIG. 1A shows a 1-atomphosphorous-containing linkage which forms the five atom repeating-unitbackbone where the morpholino rings are linked by a 1-atom phosphoamidelinkage. FIG. 1B shows a six atom repeating-unit backbone where the atomY linking the 5′ morpholino carbon to the phosphorous group may besulfur, nitrogen, carbon or, preferably, oxygen. The X moiety pendantfrom the phosphorous may be any of the following: fluorine; an alkyl orsubstituted alkyl; an alkoxy or substituted alkoxy; a thioalkoxy orsubstituted thioalkoxy; or, an unsubstituted, monosubstituted, ordisubstituted nitrogen, including cyclic structures. FIGS. 1C-D show7-atom unit-length backbones. In FIG. 1C the X moiety is as in StructureB of FIG. 1 and the moiety Y may be a methylene, sulfur, or preferablyoxygen. In the structure shown in FIG. 1D the X and Y moieties are as inFIG. 1B. In all subunits depicted in FIG. 1A-D, Z is O or S, and P_(i)or P_(j) is adenine, cytosine, guanine, thymine, uracil or inosine.

A “phosphoramidate” group comprises phosphorus having three attachedoxygen atoms and one attached nitrogen atom, while a“phosphorodiamidate” group (see e.g. FIGS. 1A-B) comprises phosphorushaving two attached oxygen atoms and two attached nitrogen atoms. In theuncharged or the cationic intersubunit linkages of the oligomersdescribed herein, one nitrogen is always pendant to the backbone chain.The second, nitrogen, in a phosphorodiamidate linkage, is typically thering nitrogen in a morpholino ring structure (again, see FIGS. 1A-B). Ina thiophosphoramidate or thiophosphorodiamidate linkage, one oxygenatom, typically the oxygen pendant to the backbone in the oligomersdescribed herein, is replaced with sulfur. Preferred morpholinooligonucleotides include those composed of morpholino subunit structuresof the form shown in FIG. 1B, where X═NH₂, N(CH₃)₂, or 1-piperazine orother charged group, Y═O, and Z═O. An oligonucleotide compound havingthis backbone structure is generally referred to herein as a “PMO”(phosphorodiamidate morpholino oligonucleotide), where “P-PMO” refers toa peptide-conjugated PMO, “PMO+” refers to a PMO with charged backbonelinkages, and “P-PMO+” refers to a peptide-conjugated PMO havingcationic backbone linkages.

The terms “charged”, “uncharged”, “cationic” and “anionic” as usedherein refer to the predominant state of a chemical moiety atnear-neutral pH, e.g. about 6 to 8. Preferably, the term refers to thepredominant state of the chemical moiety at physiological pH, that is,about 7.4. Thus a cationic backbone linkage is predominantly positivelycharged at pH 7.4.

“Lower alkyl” refers to an alkyl radical of one to six carbon atoms, asexemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl, isoamyl,n-pentyl, and isopentyl. In selected embodiments, a “lower alkyl” grouphas one to four carbon atoms, or 1-2 carbon atoms; i.e. methyl or ethyl.Analogously, “lower alkenyl” refers to an alkenyl radical of two to six,preferably three or four, carbon atoms, as exemplified by allyl andbutenyl.

A “non-interfering” substituent is one that does not adversely affectthe ability of an antisense oligomer as described herein to bind to itsintended target. Such substituents include small and preferablynon-polar groups such as methyl, ethyl, methoxy, ethoxy, hydroxy, orfluoro.

As used herein, an oligonucleotide has (or is modified to have) aspecified percentage of cationic linkages in its backbone linkages if atleast that percentage of phosphorus-containing backbone linkages arecationic, i.e., substantially positively charged at physiological pH,where substantially all of the remainder backbone linkages are unchargedat physiological pH. Thus, a 16mer oligonucleotide containing between20% and 50% cationic linkages would include between 3-8 cationiclinkages. Preferred cationic backbone linkages, and the synthesis ofmorpholino oligonucleotides containing such linkages are detailed below.

As used herein, a “nuclease-resistant” oligonucleotide molecule(oligonucleotide) is one whose backbone is not susceptible to nucleasecleavage of a phosphodiester bond.

As used herein, an antisense oligonucleotide “specifically hybridizes”to a target polynucleotide if the oligonucleotide hybridizes to thetarget under physiological conditions, with a Tm greater than 37° C. Aswill be seen below, the antisense oligonucleotides of the claimedsubject matter have a preferred Tm values with respect to their targetmRNAs of at least 45° C., typically between 50°-60° C. or greater.

The “Tm” of an oligonucleotide compound, with respect to its targetmRNA, is the temperature at which 50% of a target sequence hybridizes toa complementary polynucleotide. Tm is determined under standardconditions in physiological saline, as described, for example, in MiyadaC. G. and Wallace R. B. 1987. Oligonucleotide hybridization techniques.Methods Enzymol. 154:94-107.

Polynucleotides are described as “complementary” to one another whenhybridization occurs in an antiparallel configuration between twosingle-stranded polynucleotides. A double-stranded polynucleotide can be“complementary” to another polynucleotide, if hybridization can occurbetween one of the strands of the first polynucleotide and the second.Complementarity (the degree that one polynucleotide is complementarywith another) is quantifiable in terms of the proportion of bases inopposing strands that are expected to form hydrogen bonds with eachother, according to generally accepted base-pairing rules.

As used herein, a first sequence is an “antisense sequence” with respectto a second sequence, typically an RNA sequence, if a polynucleotidewhose sequence is the first sequence specifically binds to, orspecifically hybridizes with, the second polynucleotide sequence underphysiological conditions.

As used herein, a “base-specific intracellular binding event involving atarget RNA” refers to the sequence specific binding of anoligonucleotide to a target RNA sequence inside a cell. For example, asingle-stranded polynucleotide can specifically bind to asingle-stranded polynucleotide that is complementary in sequence.

As used herein, “essential bacterial genes” are those genes whoseproducts play an essential role in an organism's functional repertoireas determined using genetic footprinting or other comparable techniquesto identify gene essentiality.

An agent is “actively taken up by bacterial cells” when the agent canenter the cell by a mechanism other than passive diffusion across thecell membrane. The agent may be transported, for example, by “activetransport”, referring to transport of agents across a mammalian cellmembrane by e.g. an ATP-dependent transport mechanism, or by“facilitated transport”, referring to transport of antisense agentsacross the cell membrane by a transport mechanism that requires bindingof the agent to a transport protein, which then facilitates passage ofthe bound agent across the membrane.

As used herein, the terms “modulating expression” and “antisenseactivity” relative to an oligonucleotide refers to the ability of anantisense oligonucleotide to either enhance or reduce the expression ofa given protein by interfering with the expression, or translation ofRNA. In the case of reduced protein expression, the antisenseoligonucleotide may directly block expression of a given gene, orcontribute to the accelerated breakdown of the RNA transcribed from thatgene.

As used herein, the term “inhibiting bacterial growth, refers toblocking or inhibiting replication and/or reducing the rate ofreplication of bacterial cells in a given environment, for example, inan infective mammalian host.

As used herein, the term “pathogenic bacterium,” or “pathogenicbacteria,” or “pathogenic bacterial cells,” refers to bacterial cellscapable of infecting and causing disease in a mammalian host, as well asproducing infection-related symptoms in the infected host, such as feveror other signs of inflammation, intestinal symptoms, respiratorysymptoms, dehydration, and the like.

As used herein, the terms “Gram-negative pathogenic bacteria” or“Gram-negative bacteria” refer to the phylum of proteobacteria, whichhave an outer membrane composed largely of lipopolysaccharides. Allproteobacteria are gram negative, and include, but are not limited toEscherichia coli, Salmonella, other Enterobacteriaceae, Pseudomonas,Burkholderi, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio,acetic acid bacteria, and Legionella. Other notable groups of gramnegative bacteria include Haemophilus influenzae, the cyanobacteria,spirochaetes, green sulfur and green non-sulfur bacteria. The pathogeniccapability of gram negative bacteria is usually associated withcomponents of the bacterial cell wall, in particular thelipopolysaccharide (also known as LPS or endotoxin) layer.

As used herein, the terms “Gram-positive pathogenic bacteria” or“Gram-positive bacteria” refer to those bacteria that are stained darkblue or violet by Gram staining, in contrast to Gram-negative bacteria,which cannot retain the stain, instead taking up the counterstain andappearing red or pink. The stain is caused by a high amount ofpeptidoglycan in the cell wall, which typically, but not always, lacksthe secondary membrane and lipopolysaccharide layer found inGram-negative bacteria.

Gram-positive bacteria include many well-known genera such as Bacillus,Listeria, Staphylococcus, Streptococcus, Enterococcus, and Clostridium.It has also been expanded to include the Mollicutes, and bacteria suchas Mycoplasma, which lack cell walls and so cannot be stained by Gram,but are derived from such forms.

As used herein, “effective amount” or “therapeutically effective amount”or “growth-inhibiting amount” relative to an antisense oligonucleotiderefers to the amount of antisense oligonucleotide administered to amammalian subject, either as a single dose or as part of a series ofdoses and which is effective to inhibit bacterial replication in aninfected host, by inhibiting translation of a selected bacterial targetnucleic acid sequence. The ability to block or inhibit bacterialreplication in an infected host may be evidenced by a reduction ininfection-related symptoms.

As used herein “treatment” of an individual or a cell is any type ofintervention used in an attempt to alter the natural course of theindividual or cell. Treatment includes, but is not limited to,administration of e.g., a pharmaceutical composition, and may beperformed either prophylactically, or subsequent to the initiation of apathologic event or contact with an etiologic agent.

II. Constructing the Antisense Oligonucleotide

Examples of morpholino oligonucleotides having phosphorus-containingbackbone linkages are illustrated in FIGS. 1A-1D. Especially preferredis a phosphorodiamidate-linked morpholino oligonucleotide such as shownin FIG. 1B, which is modified, in accordance with one aspect of thepresent invention, to contain positively charged groups at 10%-80%,preferably 20%-50% of its backbone linkages. Morpholino oligonucleotideswith uncharged backbone linkages, including antisense oligonucleotides,are detailed, for example, in co-owned U.S. Pat. Nos. 5,698,685,5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185, 444, 5,521,063, and5,506,337, all of which are expressly incorporated by reference herein.

Important properties of the morpholino-based subunits include: 1) theability to be linked in a oligomeric form by stable, uncharged orpositively charged backbone linkages; 2) the ability to support anucleotide base (e.g. adenine, cytosine, guanine, thymidine, uracil andinosine) such that the polymer formed can hybridize with acomplementary-base target nucleic acid, including target RNA, Tm valuesabove about 45° C. in relatively short oligonucleotides (e.g., 10-15bases); 3) the ability of the oligonucleotide to be actively orpassively transported into bacterial cells; and 4) the ability of theoligonucleotide: and an antisense:RNA heteroduplex to resist RNAse andRNaseH degradation, respectively.

Exemplary backbone structures for antisense oligonucleotides of theclaimed subject matter include the morpholino subunit types shown inFIGS. 1A-1D, each linked by an uncharged or positively charged,phosphorus-containing subunit linkage. FIG. 1A shows aphosphorus-containing linkage which forms the five atom repeating-unitbackbone, where the morpholino rings are linked by a 1-atom phosphoamidelinkage. FIG. 1B shows a linkage which produces a 6-atom repeating-unitbackbone. In this structure, the atom Y linking the 5′ morpholino carbonto the phosphorus group may be sulfur, nitrogen, carbon or, preferably,oxygen. The X moiety pendant from the phosphorus may be fluorine, analkyl or substituted alkyl, an alkoxy or substituted alkoxy, athioalkoxy or substituted thioalkoxy, or unsubstituted, monosubstituted,or disubstituted nitrogen, including cyclic structures, such asmorpholines or piperidines. Alkyl, alkoxy and thioalkoxy preferablyinclude 1-6 carbon atoms. The Z moieties are sulfur or oxygen, and arepreferably oxygen.

The linkages shown in FIGS. 1C and 1D are designed for 7-atomunit-length backbones. In Structure 1C, the X moiety is as in Structure1B, and the moiety Y may be methylene, sulfur, or, preferably, oxygen.In Structure 1D, the X and Y moieties are as in Structure 1B.Particularly preferred morpholino oligonucleotides include thosecomposed of morpholino subunit structures of the form shown in FIG. 1B,where X═NH₂, N(CH₃)₂, or 1-piperazine or other charged group, Y═O, andZ═O.

As noted above, the substantially uncharged oligonucleotide may bemodified, in accordance with an aspect of the invention, to includecharged linkages, e.g. up to about 1 per every 2-5 uncharged linkages,such as about 4-5 per every 10 uncharged linkages. Optimal improvementin antisense activity may be seen when about half of the backbonelinkages are cationic. Suboptimal enhancement is typically seen with asmall number e.g., 10-20% cationic linkages, and where the number ofcationic linkages are in the range 50-80%, and typically above about6-%, the sequence specificity of the antisense binding to its target maybe compromised or lost.

Additional experiments conducted in support of the present inventionindicate that the enhancement seen with added cationic backbone chargesmay, in some cases, be further enhanced by distributing the bulk of thecharges close of the “center-region” backbone linkages of the antisenseoligonucleotide, e.g., in a 20mer oligonucleotide with 8 cationicbackbone linkages, having at least 70% of these charged linkageslocalized in the 10 centermost linkages.

The antisense compounds can be prepared by stepwise solid-phasesynthesis, employing methods detailed in the references cited above, andbelow with respect to the synthesis of oligonucleotides having a mixtureor uncharged and cationic backbone linkages. In some cases, it may bedesirable to add additional chemical moieties to the antisense compound,e.g. to enhance pharmacokinetics or to facilitate capture or detectionof the compound. Such a moiety may be covalently attached, typically toa terminus of the oligomer, according to standard synthetic methods. Forexample, addition of a polyethyleneglycol moiety or other hydrophilicpolymer, e.g., one having 10-100 monomeric subunits, may be useful inenhancing solubility. One or more charged groups, e.g., anionic chargedgroups such as an organic acid, may enhance cell uptake. A reportermoiety, such as fluorescein or a radiolabeled group, may be attached forpurposes of detection. Alternatively, the reporter label attached to theoligomer may be a ligand, such as an antigen or biotin, capable ofbinding a labeled antibody or streptavidin. In selecting a moiety forattachment or modification of an antisense oligomer, it is generally ofcourse desirable to select chemical compounds of groups that arebiocompatible and likely to be tolerated by a subject withoutundesirable side effects.

A. Oligomers with Cationic Intersubunit Linkages

This section considers the structures and synthesis of antisenseoligonucleotides that are modified, in accordance with one aspect of theinvention, to include multiple cationic charges in the backbonelinkages. The intersubunit linkages, both uncharged and cationic,preferably are phosphorus-containing linkages, having the structure:

where

W is S or O, and is preferably O,

X═NR¹R² or OR⁶,

Y═O or NR⁷,

-   -   and each said linkage in the oligomer is selected from:    -   (a) uncharged linkage (a), where each of R¹, R², R⁶ and R⁷ is        independently selected from hydrogen and lower alkyl;    -   (b1) cationic linkage (b1), where X═NR¹R² and Y═O, and NR¹R²        represents an optionally substituted piperazino group, such that        R¹R²═—CHRCHRN(R³)(R⁴)CHRCHR—, where    -   each R is independently H or CH₃,    -   R⁴ is H, CH₃, or an electron pair, and    -   R³ is selected from H, lower alkyl, e.g. CH₃, C(═NH)NH₂,        Z-L-NHC(═NH)NH₂, and [C(O)CHR′NH]_(m)H, where: Z is C(O) or a        direct bond, L is an optional linker up to 18 atoms in length,        preferably up to 12 atoms, and more preferably up to 8 atoms in        length, having bonds selected from alkyl, alkoxy, and        alkylamino, R′ is a side chain of a naturally occurring amino        acid or a one- or two-carbon homolog thereof, and m is 1 to 6,        preferably 1 to 4;    -   (b2) cationic linkage (b2), where X═NR¹R² and Y═O, R¹═H or CH₃,        and R²=LNR³R⁴R⁵, where L, R³, and R⁴ are as defined above, and        R⁵ is H, lower alkyl, or lower (alkoxy)alkyl; and    -   (b3) cationic linkage (b3), where Y═NR⁷ and X═OR⁶, and        R=LNR³R⁴R⁵, where L, R³, R⁴ and R⁵ are as defined above, and R⁶        is H or lower alkyl;    -   and at least one said linkage is selected from cationic linkages        (b1), (b2), and (b3).

Preferably, the oligomer includes at least two consecutive linkages oftype (a) (i.e. uncharged linkages). In further embodiments, at least 5%of the linkages in the oligomer are cationic linkages (i.e. type (b1),(b2), or (b3)); for example, 10% to 60%, and preferably 20-50% linkagesmay be cationic linkages.

In one embodiment, at least one linkage is of type (b1), where,preferably, each R is H, R⁴ is H, CH₃, or an electron pair, and R³ isselected from H, lower alkyl, e.g. CH₃, C(═NH)NH₂, andC(O)-L-NHC(═NH)NH₂. The latter two embodiments of R³ provide a guanidinomoiety, either attached directly to the piperazine ring, or pendant to alinker group L, respectively. For ease of synthesis, the variable Z inR³ is preferably C(O) (carbonyl), as shown.

The linker group L, as noted above, contains bonds in its backboneselected from alkyl (e.g. —CH₂—CH₂—), alkoxy (—C—O—), and alkylamino(e.g. —CH₂—NH—), with the proviso that the terminal atoms in L (e.g.,those adjacent to carbonyl or nitrogen) are carbon atoms. Althoughbranched linkages (e.g. —CH₂—CHCH₃—) are possible, the linker ispreferably unbranched. In one embodiment, the linker is a hydrocarbonlinker. Such a linker may have the structure —(CH₂)_(n)—, where n is1-12, preferably 2-8, and more preferably 2-6.

The morpholino subunits have the structure:

where Pi is a base-pairing moiety, and the linkages depicted aboveconnect the nitrogen atom of (i) to the 5′ carbon of an adjacentsubunit. The base-pairing moieties Pi may be the same or different, andare generally designed to provide a sequence which binds to a targetnucleic acid.

The use of embodiments of linkage types (b1), (b2) and (b3) above tolink morpholino subunits may be illustrated graphically as follows:

Preferably, all cationic linkages in the oligomer are of the same type;i.e. all of type (b1), all of type (b2), or all of type (b3).

In further embodiments, the cationic linkages are selected from linkages(b1′) and (b1″) as shown below, where (b1″) is referred to herein as a“Pip” linkage and (b1″) is referred to herein as a “GuX” linkage:

In the structures above, W is S or O, and is preferably O; each of R¹and R² is independently selected from hydrogen and lower alkyl, and ispreferably methyl; and A represents hydrogen or a non-interferingsubstituent on one or more carbon atoms in (b1′) and (b1″). Preferably,the ring carbons in the piperazine ring are unsubstituted; however, theymay include non-interfering substituents, such as methyl or fluorine.Preferably, at most one or two carbon atoms is so substituted.

In further embodiments, at least 10% of the linkages are of type (b1′)or (b1″); for example, 10%-60% and preferably 20% to 50%, of thelinkages may be of type (b1′) or (b1″).

In other embodiments, the oligomer contains no linkages of the type(b1′) above. Alternatively, the oligomer contains no linkages of type(b1) where each R is H, R³ is H or CH₃, and R⁴ is H, CH₃, or an electronpair.

The morpholino subunits may also be linked by non-phosphorus-basedintersubunit linkages, as described further below, where at least onelinkage is modified with a pendant cationic group as described above.

Other oligonucleotide analog linkages which are uncharged in theirunmodified state but which could also bear a pendant amine substituentcould be used. For example, a 5′ nitrogen atom on a morpholino ringcould be employed in a sulfamide linkage (see e.g. FIG. 2G) or a urealinkage (where phosphorus is replaced with carbon or sulfur,respectively) and modified in a manner analogous to the ₅′-nitrogen atomin structure (b3) above.

Oligomers having any number of cationic linkages are provided, includingfully cationic-linked oligomers. Preferably, however, the oligomers arepartially charged, having, for example, 10%-80%. In preferredembodiments, about 10% to 60%, and preferably 20% to 50% of the linkagesare cationic.

In one embodiment, the cationic linkages are interspersed along thebackbone. The partially charged oligomers preferably contain at leasttwo consecutive uncharged linkages; that is, the oligomer preferablydoes not have a strictly alternating pattern along its entire length.

Also considered are oligomers having blocks of cationic linkages andblocks of uncharged linkages; for example, a central block of unchargedlinkages may be flanked by blocks of cationic linkages, or vice versa.In one embodiment, the oligomer has approximately equal-length 5′, 3′and center regions, and the percentage of cationic linkages in thecenter region is greater than about 50%, preferably greater than about70%.

Oligomers for use in antisense applications generally range in lengthfrom about 10 to about 40 subunits, more preferably about 10 to 25subunits, and typically 10-20 bases. For example, an oligomer of theinvention having 19-20 subunits, a useful length for an antisenseoligomer, may ideally have two to ten, e.g. four to eight, cationiclinkages, and the remainder uncharged linkages. An oligomer having 14-15subunits may ideally have two to five, e.g. 3 or 7, cationic linkagesand the remainder uncharged linkages.

Each morpholino ring structure supports a base pairing moiety, to form asequence of base pairing moieties which is typically designed tohybridize to a selected antisense target in a cell or in a subject beingtreated. The base pairing moiety may be a purine or pyrimidine found innative DNA or RNA (A, G, C, T, or U) or an analog, such as hypoxanthine(the base component of the nucleoside inosine) or 5-methyl cytosine.

B. Carrier Peptides

This section considers the structures and synthesis of antisenseoligonucleotides that are modified, in accordance with another aspect ofthe invention, to include an arginine-rich carrier peptide whichenhances the antibacterial activity of the oligonucleotide, at least inpart, by enhances transport of the oligomer into bacterial cells. Thetransport moiety is preferably attached to a terminus of the oligomer,as shown, for example, in FIGS. 2P-2Q.

Preferably, the transport moiety comprises 6 to 16, preferably 8-14,amino acids and is composed of subsequences selected from the grouprepresented by (X′Y′X′), (X′Y′), (X′Z′), and (X′Z′Z′),

where

(a) each X′ subunit independently represents lysine, arginine or anarginine analog, said analog being a cationic α-amino acid comprising aside chain of the structure R¹N═C(NH₂)R², where R¹ is H or R; R² is R,NH₂, NHR, or NR₂, where R is lower alkyl or lower alkenyl and mayfurther include oxygen or nitrogen; R¹ and R² may together form a ring;and the side chain is linked to said amino acid via R¹ or R²;

(b) each Y′ subunit independently represents a neutral linear amino acid—C(O)—(CHR)_(m)—NH—, where m is 1 to 7 and each R is independently H ormethyl; and

(c) each Z′ subunit independently represents an α-amino acid having aneutral aralkyl side chain.

As used herein, a carrier protein is “composed of the subsequencesselected from the group represented by X′Y′X′, X′Y′, X′Z′Z′ and X′Z′” ifsubstantially all of its amino acids can be represented by anon-overlapping series of the subsequences, or positional variationsthereof, e.g., (X′X′Y′)_(n), (X′Y′X′)_(n), (Y′X′X′)_(n), (Y′X′)_(n),(X′Y′)(X′X′Y′)(X′Y′)(X′X′Y′), (X′Y′)_(n)(X′X′Y′)_(m), (X′FF)_(n) or(FFX′)_(n). The protein may accommodate a small number, e.g., 1-3, ofneutral amino acids other than Y or Z.

In selected embodiments, the peptide comprises a sequence which consistsof at least two, or at least three, repeats of a single subsequenceselected from (X′Y′X′), (X′Y′), (X′Z′), and (X′Z′Z′). For example, thepeptide may comprise a sequence represented by one of (X′Y′X′)_(p),(X′Y′)_(m), and (X′Z′Z′)_(p), where p is 2 to 5 and m is 2 to 8.

In selected embodiments, for each X′, the side chain moiety isindependently selected from the group consisting of guanidyl(HN═C(NH₂)NH—), amidinyl (HN═C(NH₂)C<), 2-aminodihydropyrimidyl,2-aminotetrahydropyrimidyl, 2-aminopyridinyl, and 2-aminopyrimidonyl,and it is preferably selected from guanidyl and amidinyl.

In preferred embodiments, for each X′, the side chain moiety isguanidyl, as in the amino acid subunit arginine (Arg). In furtherembodiments, each Y′ is —CO—(CH₂)_(m)—R—NH—, where m is 1 to 7 and R isH. For example, when m is 5 and R is H, Y′ is a 6-aminohexanoic acidsubunit, abbreviated herein as Ahx; when m is 2 and R is H, Y′ is a3-alanine subunit.

The aralkyl side chain of the Z′ subunit is preferably benzyl (—CH₂C₆H₆)or phenethyl (—CH₂CH₂C₆H₆), which are preferably not further substitutedbut may include a non-interfering substituent as defined herein.Preferably, the side chain is benzyl (—CH₂C₆H₆), such that each Z′ isphenylalanine (F).

One type of preferred peptides of this type include those comprisingarginine dimers alternating with single Y′ subunits, where Y′ ispreferably Ahx. Examples include peptides having the formula (RY′R)₄ orthe formula (RRY′)₄, where Y′ is preferably Ahx. In one embodiment, Y′is a 6-aminohexanoic acid subunit, R is arginine, and p is 4. In afurther embodiment, the peptide comprises a sequence represented by(X′Z′Z′)_(p), where R is arginine, each Z′ is phenylalanine, and p is 3or 4.

The conjugated peptide is preferably linked to a terminus of theoligomer via a linker Ahx-B, where Ahx is a 6-aminohexanoic acid subunitand B is a β-alanine subunit. The Y′ subunits are either contiguous, inthat no X′ subunits intervene between Y′ subunits, or interspersedsingly between X′ subunits. However, the linking subunit may be betweenY′ subunits. In one embodiment, the Y′ subunits are at a terminus of thetransporter; in other embodiments, they are flanked by X′ subunits.

In addition to enhanced uptake, the carrier peptide may help tostabilize a heteroduplex between an antisense oligomer and its targetnucleic acid sequence, by virtue of electrostatic interaction betweenthe positively charged transport moiety and the negatively chargednucleic acid. The number of charged subunits in the transporter is lessthan 14, as noted above, and preferably between 6 and 11, since too higha number of charged subunits may lead to a reduction in sequencespecificity.

Exemplary peptide transporters, including linkers (B or AhxB) are givenbelow:

SEQ Sequence ID Peptide (N-terminal to C-terminal) NO: (RRAhx)₄BRRAhxRRAhxRRAhxRRAhxB  99 (RAhxR)₄AhxB RAhxRRAhxRRAhxRRAhxRAhxB 100(AhxRR)₄AhxB AhxRRAhxRRAhxRRAhxRRAhxB 101 (RAhx)₆BRAhxRAhxRAhxRAhxRAhxRAhxB 102 (RAhx)₆B RahxRAhxRAhxRAhxRAhxRAhxRAhxB 103(RAhxR)₃AhxB RAhxRRAhxRRAhxRAhxB 104 (RAhxRRBR)₂AhxBRAhxRRBRRAhxRRBRAhxB 105 ((RB)₃RAhx)₂B RBRBRBRAhxRBRBRBRAhxB 106

C. Preparation of Oligomers Having Cationic Intersubunit Linkages

FIGS. 2A through 2R illustrate the preparation of morpholino subunitshaving suitably protected base-pairing groups, and the conversion ofthese subunits into morpholino oligomers having cationic linkages.Further experimental detail is provided in Materials and Methods, below.The charged-linkage subunits can be used in standard stepwise oligomersynthesis, as described, for morpholino oligomers, in U.S. Pat. No.5,185,444 or in Summerton and Weller, 1997 (cited above).

FIG. 2A shows representative morpholino subunits 1a-e with base-pairingmoieties Pi of A, C, G, T, and I. These subunits can be prepared fromthe corresponding ribonucleosides as illustrated in FIG. 2B anddescribed in Example 1. Suitable protecting groups are used for thenucleoside bases, where necessary; for example, benzoyl for adenine andcytosine, phenylacetyl for guanine, and pivaloyloxymethyl forhypoxanthine (I). The pivaloyloxymethyl group can be introduced onto theN1 position of the hypoxanthine heterocyclic base as shown in FIG. 2B.Although an unprotected hypoxanthine subunit, as in if, may be employed,yields in activation reactions are far superior when the base isprotected.

Treatment of the 5′-hydroxy (1) with a reactive acid chloride, such asN,N-dimethylphosphoramidodichloridate (4), provides type (a) (unchargedlinkage) activated subunits 5a-e, as shown in FIG. 2C and described inExample 2. Although the unprotected hypoxanthine containing subunit, asin if, may be employed, yields in activation reactions are far superiorwhen the base is protected.

FIG. 2C also illustrates the use of alternate reactive acid chlorides,such as 6a, to convert 5′-hydroxy subunits 1a-e into type (b1) (chargedlinkage) activated subunits 7a-e.

Similarly, an acyclic reactive acid chloride, such as 8a, can be used toconvert 5′-hydroxy subunits 1a-e into type (b2) (charged linkage)activated subunits 9a-e. These charged-linkage subunits may beincorporated into phosphorodiamidate-linked morpholino oligomers and,upon treatment with the usual reagents that remove the base protectinggroups, preferably ammonia, produce oligomers containing type (b1) and(b2) cationic phosphorodiamidate linkages.

A schematic of a synthetic pathway that can be used to make morpholinosubunits containing the (1-piperazino) phosphinylideneoxy linkage (typeb1′; “Pip”) is shown in FIG. 2D and described in Example 3. Reaction ofpiperazine and trityl chloride 10 gives trityl piperazine, which can beisolated as the succinate salt 11. Reaction with ethyl trifluoroacetate13a in the presence of a weak base, e.g. diisopropylethylamine, provides1-trifluoroacetyl-4-trityl piperazine 14, which upon treatment with HClprovide the detritylated salt 15 in good yield. Introduction of thedichlorophosphoryl moiety on the free eing nitrogen was performed withphosphorus oxychloride in toluene, yielding the piperazine-P(O)Cl₂moiety 6a. This reagent can be reacted with 5′-hydroxy morpholinosubunits to produce activated subunits containing the protected(1-piperazino) phosphinylideneoxy linkage, which can be incorporatedinto oligomers using the oligomer synthesis protocol below.

Selectively protected acyclic amines, suitable for incorporation intomorpholino subunits for the preparation of type (b2) cationic linkages,may be prepared by methods analogous to that described and illustratedfor the cyclic amines; see Example 4. Alternatively, treatment of asolution of a diamine with 1.6 equivalents of the reactive ester 13a-dprovides a solution with <5% of the free diamino species. The solutionwas used directly for activation with POCl₃ and activation of themorpholino subunits 1a-e. A person skilled in the art would find itpossible to prepare oligomers with more complex cationic sides chainsusing the methods above.

Subunits for the introduction of type (b3) cationic linkages, i.e.having a nitrogen at the 5′-position, into oligomers may be prepared, asshown in FIG. 2E and described in Example 5, by oxidation of amorpholino subunit to the corresponding aldehyde (16a-e) and reductiveamination with a suitably protected diamine, which affords arepresentative 5′-aminomorpholino subunit 20a-e. It is often preferableto isolate the amine as the 9-fluorenylmethyloxycarbonyl (FMOC)derivative 21a-e following treatment with FMOC chloride. The free aminecan be easily regenerated when needed by treatment with triethylamine or1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Activation of the amine withethyl phosphorodichloridate gives type (b3) activated subunits 22a-e,which can be incorporated into oligomers in the same manner as type (a),(b1) and (b2) subunits.

A method for the preparation of variants of 22a-e, containing variousside chains on the 5′-nitrogen, involves alkylation of an activated5′-morpholino subunit with suitably protected amines. As shown in FIG.2F for two examples, and described in Example 6, hexamethylene diaminewas first protected, then reacted with 5′-O-p-toluenesulfonated subunit23a-e. Using the methods in FIGS. 2E and 2F and in the correspondingExamples, a person skilled in the art could prepare a wide range of5′-amino substituted subunits suitable for incorporation into cationicmorpholino oligomers.

As noted above, cationic linkages may also be prepared fromnon-phosphorus-containing linkages. For example, subunits capable ofproviding sulfonamide linkages with pendant cationic groups may beintroduced from the amine used in (b3) type linkages, as shown in FIG.2G and described in Example 7. Reaction of the aminated subunits withsulfur-trioxide/pyridine in N,N-dimethylformamide containingtriethylamine provides a sulfamic acid that was treated with phosgene indichloromethane containing pyridine to give the activated sulfamoylchloride containing subunit.

Morpholino oligomers can be prepared from such subunits in a stepwisemanner on a solid support, preferably an aminomethyl polystyrene solidsupport, e.g. as described in U.S. Pat. No. 5,185,444 or in Summertonand Weller, 1997 (cited above). The resin is preferably modified byreaction with a disulfide “anchor”, which allows production of themorpholino oligomer on the support and facile release upon treatmentwith a thiol, as shown in FIG. 2H and described in Example 8.

In some cases it is advantageous to introduce a triethylene glycolcontaining moiety (“tail”) which increases aqueous solubility of themorpholino oligomers. One method for accomplishing this is illustratedin FIG. 2I and described in Example 9.

In a typical synthesis, the disulfide anchor 34 is reacted as shown inFIG. 2J with aminomethylpolystyrene resin in 1-methyl-2-pyrrolidinone(NMP) to give resin-anchor 39, suitable for incorporation of activatedsubunits. Optionally, the Tail moiety can be introduced onto the5′-terminus of the oligomer by reaction of the disulfide anchor-resinwith 38 to produce Tail-resin 40. Use of resin 40 will cause theHOCH₂CH₂OCH₂CH₂OCH₂CH₂OC(O) group (=EG3) to become attached to the5′-terminus of the oligomer.

The activated subunits, containing the appropriate intersubunit linkagetype, are introduced stepwise by solid phase synthesis on resin 39containing anchor or, optionally, the Tail resin 40. A cycle of solidphase synthesis performed using an automated synthesizer consists ofwashing the resin with NMP or dichloromethane (DCM), followed bytreatment with 11% cyanoacetic acid in 20% acetonitrile/DCM (v/v). Afterneutralization with a 5% solution of diisopropylethylamine (DIEA) in 20%isopropanol/DCM, the resin is reacted with a 0.2 M solution of theactivated subunit in 1,3-dimethyl-2-imidazolidinone (DMI) (or Tail inNMP) containing 0.4 M 4-ethylmorpholine. After washing withneutralization solution, the cycle may be repeated to introduce the nextsubunit. Optionally, following the final subunit addition, the tritylgroup at the end of the resin is removed and methoxytrityl chlorideintroduced to prepare the 3′-methoxytritylated oligomer. The more labilemethoxytrityl species provides benefit in the aqueous detritylation stepwhich follows “trityl-ON/trityl-OFF” purification of the crudeoligomers.

The reactor design used for the preparation of the bulk resins 39 and 40was employed for larger scale synthesis of morpholino oligomers. On thelarge scale, the detritylation steps performed when phosphorodiamidatelinkages had been introduced onto the resin used a solution of4-cyanopyridinium trifluoroacetate in 20% trifluoroethanol/DCM. Thisprovided less hydrolysis of the somewhat acid labile phosphorodiamidatelinkages than did carboxylic acid based detritylation reagents.Additionally, the use of doubly protected G subunit was found to beadvantageous. FIG. 2K illustrates synthesis of the N2,O6-protected Gspecies 46 that was employed.

The synthesized oligomers were released from the solid support bytreatment with a solution of 1,4-dithiothreitol and triethylamine inNMP. The solution was treated with concentrated ammonia and held at 45°C. The mixture was sealed in a pressure vessel and heated at 45° C. for16-24 hours. The solution was diluted with 0.28% aqueous ammonia andpassed through ion exchange resin to capture the crude methoxytritylatedoligomer. The product was eluted with a salt gradient to recover thelater-eluting, methoxytrityl or trityl containing product and theproduct containing fractions pooled. For preparation of 3′-unsubstituted(3′-H) oligomers requiring no further modification, the solution wastreated with acid to pH=2.5 to demethoxytritylate the oligomer. Thedemethoxytritylation mixture was immediately neutralized withconcentrated ammonia, and the solution passed through reversed phaseresin. The product was recovered by elution with 45% acetonitrile/0.28%aqueous ammonia and isolated as a white powder after lyophilization.Further purification of the product may be performed on cation exchangeresins as described in the methods section. Alternatively, it wasadvantageous to retain the 3′-methoxytrityl/trityl group in order toperform modification of the backbone amine moieties independent of the3′-terminus of the oligomer, as described below. It this case, the aboveprocedure was followed except that the aqueous acid treatment wasomitted.

Amine groups introduced into a morpholino oligomer as part of cationiclinkages may be further modified. This concept allows an oligomer to beconstructed from a relatively simple modified subunit, but withfunctionality sufficient to allow the introduction of complex moietiesin any location along the backbone of the morpholino oligomers.

Note that, for reasons of synthesis, the 5′ terminal linkage of anoligomer does not typically comprise a linkage of type (b1) describedherein. As shown, for example, in FIGS. 2P-2Q, the preferred stepwiseresin-supported synthesis of the oligomers provides a piperazine ring onthe phosphorus atom at the 5′ terminus; the presence of a secondpiperazine ring on the phosphorus would be constrained for stericreasons.

An important modification is the incorporation of guanidinium groupsinto the oligomer. This may be done in two ways. In the first, the aminemoiety on the backbone of the oligomer was directly converted into aguanidinium species by reaction with 1H-pyrazole-1-carboxamidinehydrochloride (M S Bernatowicz, Y Wu, G R Matsueda, J. Org. Chem., 1992,57(8), 2497-2502) in sodium carbonate buffered aqueous solution, as inFIG. 2L, which also shows the EG3 Tail at the 5′-terminus. In thesecond, a substance containing both carboxyl and guanidinium groups,e.g., 6-guanidinohexanoic acid was activated with2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate(HBTU) and reacted with the amine containing oligomer (FIG. 2M). In asimilar fashion, 4-guanidinobutanoic acid, 3-guanidinopropanoic acid,and guanidinoacetic acid may be introduced. In a hybrid of theseapproaches, the amine moiety was reacted with a protected FMOC aminoacid, e.g., FMOC 6-aminohexanoic acid to introduce a protected primaryamine containing side chain, which after treatment with ammonia toremove the FMOC group was guanylated as above. Fully guanylated specieswere separated from partially guanylated oligomers by cationchromatography at the appropriate pH.

The termini of the oligomer can also eb substituted with guanidiniummoieties by these methods, as illustrated in FIG. 2N, which also shows arepresentative oligomer created from resin 39, without addition of thePEG Tail.

Another modification of note is the incorporation of peptides along thebackbone. Small peptides are readily available from commercial sources,for example, Bachem Calif., Inc. 3132 Kashiwa Street Torrance, Calif.90505 USA, and AnaSpec, Inc. 2149 O'Toole Ave., San Jose, Calif. 95131.The incorporation of the peptide followed classic2-(1-H-benzotriazol-1-yl)-1,1,3,3; tetramethylaminiumhexafluorophosphate (HBTU) chemistry, as illustrated in FIG. 2O.Guanidinium groups on the oligomer or peptide do not interfere.

Oligomers may also be conjugated at the 3′-terminus to arginine richpeptides, useful to enhance delivery of the products into cells. In thiscase, protection of primary and secondary amine moieties along thebackbone of 3′-methoxtritylated/tritylated oligomers was performed bytrifluoroacetylation, as shown in FIG. 2P. The terminal methoxytritylgroup was removed and the peptide conjugated using HBTU. The conjugationreaction was worked up by treatment with ammonia to remove thetrifluoroacetyl groups. The conjugate was purified by cation exchangechromatography. When the backbone amine functions are fully guanylated,the peptide may be introduced without interference from these sidechains, as shown in FIG. 2Q.

D. Antibacterial Antisense Oligonucleotides

In addition to the structural features described above, the antisensecompound of the claimed subject matter contains no more than about 30,preferably no more than about 20 nucleotide bases, and has a targetingnucleic acid sequence (the sequence which is complementary to the targetsequence) of no fewer than 10 contiguous bases. In one generalembodiment, the targeting sequence is complementary to a target sequencecontaining or within 15 bases, in a downstream direction, of thetranslational start codon of a bacterial mRNA that encodes a bacterialprotein essential for bacterial replication. In another generalembodiment, the targeting sequence is a region of the 16S or 23S RNA ofthe 30S ribosome subunit.

The compound has a T_(m), when hybridized with the target sequence, ofat least about 45° C., typically between about 50° to 60° C., althoughthe Tm may be higher, e.g., 65° C. The selection of bacterial targets,and bacterial mRNA target sequences and complementary targetingsequences are considered in below.

The antisense morpholino oligonucleotide has enhanced antibacterialactivity by virtue of its being conjugated to an 8 to 14 residuearginine-rich peptide at either the 5′- or 3′-end of the oligonucleotideand/or by virtue of containing between positively charged groups in atleast 10%-80%, preferably 20%-50% of its backbone linkages. As will beseen below, each of these two modifications may enhance antibacterialactivity by 10-fold over the unmodified oligonucleotide, and the twomodifications together may enhance activity by 100-1,000 fold or moreover the unmodified oligonucleotide, allowing a reduction in the amountof compound needed for effective antibacterial activity by 100-1,000fold or more.

C1. Bacterial Targets

Escherichia coli (E. coli) is a Gram-negative bacterium that is part ofthe normal flora of the gastrointestinal tract. There are hundreds ofstrains of E. coli, most of which are harmless and live in thegastrointestinal tract of healthy humans and animals. Currently, thereare four recognized classes of enterovirulent E. coli (the “EEC group”)that cause gastroenteritis in humans. Among these are theenteropathogenic (EPEC) strains and those whose virulence mechanism isrelated to the excretion of typical E. coli enterotoxins. Such strainsof E. coli can cause various diseases including those associated withinfection of the gastrointestinal tract and urinary tract, septicemia,pneumonia, and meningitis. Antibiotics are not effective against somestrains and do not necessarily prevent recurrence of infection.

For example, E. coli strain 0157:H7 is estimated to cause 10,000 to20,000 cases of infection in the United States annually (Federal Centersfor Disease Control and Prevention). Hemorrhagic colitis is the name ofthe acute disease caused by E. coli O157:H7. Preschool children and theelderly are at the greatest risk of serious complications. E. colistrain 0157:H7 was recently reported as the cause the death of fourchildren who ate under cooked hamburgers from a fast-food restaurant inthe Pacific Northwest. (See, e.g., Jackson et al., Epidemiol. Infect.120(1):17-20, 1998.)

Exemplary sequences for enterovirulent E. coli strains include GenBankAccession Numbers AB011549, X97542, AF074613, Y11275 and AJ007716.

Salmonella thyphimurium, are Gram-negative bacteria which cause variousconditions that range clinically from localized gastrointestinalinfections, gastroenteritis (diarrhea, abdominal cramps, and fever) toenteric fevers (including typhoid fever) which are serious systemicillnesses. Salmonella infection also causes substantial losses oflivestock.

Typical of Gram-negative bacilli, the cell wall of Salmonella spp.contains a complex lipopolysaccharide (LPS) structure that is liberatedupon lysis of the cell and may function as an endotoxin, whichcontributes to the virulence of the organism.

Contaminated food is the major mode of transmission for non-typhoidalsalmonella infection, due to the fact that Salmonella survive in meatsand animal products that are not thoroughly cooked. The most commonanimal sources are chickens, turkeys, pigs, and cows; in addition tonumerous other domestic and wild animals. The epidemiology of typhoidfever and other enteric fevers caused by Salmonella spp. is associatedwith water contaminated with human feces.

Vaccines are available for typhoid fever and are partially effective;however, no vaccines are available for non-typhoidal Salmonellainfection. Non-typhoidal salmonellosis is controlled by hygienicslaughtering practices and thorough cooking and refrigeration of food.Antibiotics are indicated for systemic disease, and Ampicillin has beenused with some success. However, in patients under treatment withexcessive amounts of antibiotics, patients under treatment withimmunosuppressive drugs, following gastric surgery, and in patients withhemolytic anemia, leukemia, lymphoma, or AIDS, Salmonella infectionremains a medical problem.

Pseudomonas spp. are motile, Gram-negative rods which are clinicallyimportant because they are resistant to most antibiotics, and are amajor cause of hospital acquired (nosocomial) infections. Infection ismost common in: immunocompromised individuals, burn victims, individualson respirators, individuals with indwelling catheters, IV narcotic usersand individual with chronic pulmonary disease (e.g., cystic fibrosis).Although infection is rare in healthy individuals, it can occur at manysites and lead to urinary tract infections, sepsis, pneumonia,pharyngitis, and numerous other problems, and treatment often fails withgreater significant mortality.

Pseudomonas aeruginosa is a Gram-negative, aerobic, rod-shaped bacteriumwith unipolar motility. An opportunistic human pathogen, P. aeruginosais also an opportunistic pathogen of plants. Like other Pseudomonads, P.aeruginosa secretes a variety of pigments. Definitive clinicalidentification of P. aeruginosa can include identifying the productionof both pyocyanin and fluorescein as well as the organism's ability togrow at 42° C. P. aeruginosa is also capable of growth in diesel and jetfuel, for which it is known as a hydrocarbon utilizing microorganism (or“HUM bug”), causing microbial corrosion.

Vibrio cholera is a Gram-negative rod which infects humans and causescholera, a disease spread by poor sanitation, resulting in contaminatedwater supplies. Vibrio cholerae can colonize the human small intestine,where it produces a toxin that disrupts ion transport across the mucosa,causing diarrhea and water loss. Individuals infected with Vibriocholerae require rehydration either intravenously or orally with asolution containing electrolytes. The illness generally self-limiting;however, death can occur from dehydration and loss of essentialelectrolytes. Antibiotics such as tetracycline have been demonstrated toshorten the course of the illness, and oral vaccines are currently underdevelopment.

Neisseria gonorrhoea is a Gram-negative coccus, which is the causativeagent of the common sexually transmitted disease, gonorrhea. Neisseriagonorrhoea can vary its surface antigens, preventing development ofimmunity to reinfection. Nearly 750,000 cases of gonorrhea are reportedannually in the United States, with an estimated 750,000 additionalunreported cases annually, mostly among teenagers and young adults.Ampicillin, amoxicillin, or some type of penicillin used to berecommended for the treatment of gonorrhea. However, the incidence ofpenicillin-resistant gonorrhea is increasing, and new antibiotics givenby injection, e.g., ceftriaxone or spectinomycin, are now used to treatmost gonococcal infections.

Staphylococcus aureus is a Gram-positive coccus which normally colonizesthe human nose and is sometimes found on the skin. Staphylococcus cancause bloodstream infections, pneumonia, and surgical-site infections inthe hospital setting (i.e., nosocomial infections). Staph. aureus cancause severe food poisoning, and many strains grow in food and produceexotoxins. Staphylococcus resistance to common antibiotics, e.g.,vancomycin, has emerged in the United States and abroad as a majorpublic health challenge both in community and hospital settings.Recently, a vancomycin-resistant Staph. aureus isolate has also beenidentified in Japan.

Mycobacterium tuberculosis is a Gram positive bacterium which is thecausative agent of tuberculosis, a sometimes crippling and deadlydisease. Tuberculosis is on the rise and globally and the leading causeof death from a single infectious disease (with a current death rate ofthree million people per year). It can affect several organs of thehuman body, including the brain, the kidneys and the bones, however,tuberculosis most commonly affects the lungs.

In the United States, approximately ten million individuals are infectedwith Mycobacterium tuberculosis, as indicated by positive skin tests,with approximately 26,000 new cases of active disease each year. Theincrease in tuberculosis (TB) cases has been associated with HIV/AIDS,homelessness, drug abuse and immigration of persons with activeinfections. Current treatment programs for drug-susceptible TB involvetaking two or four drugs (e.g., isoniazid, rifampin, pyrazinamide,ethambutol or streptomycin), for a period of from six to nine months,because all of the TB germs cannot be destroyed by a single drug. Inaddition, the observation of drug-resistant and multiple drug resistantstrains of Mycobacterium tuberculosis is on the rise.

Helicobacter pylori (H. pylori) is a micro-aerophilic, Gram-negative,slow-growing, flagellated organism with a spiral or S-shaped morphologywhich infects the lining of the stomach. H. pylori is a human gastricpathogen associated with chronic superficial gastritis, peptic ulcerdisease, and chronic atrophic gastritis leading to gastricadenocarcinoma. H. pylori is one of the most common chronic bacterialinfections in humans and is found in over 90% of patients with activegastritis. Current treatment includes triple drug therapy with bismuth,metronidazole, and either tetracycline or amoxicillin which eradicatesH. pylori in most cases. Problems with triple therapy include patientcompliance, side effects, and metronidazole resistance. Alternateregimens of dual therapy which show promise are amoxicillin plusmetronidazole or omeprazole plus amoxicillin.

Streptococcus pneumoniae is a Gram-positive coccus and one of the mostcommon causes of bacterial pneumonia as well as middle ear infections(otitis media) and meningitis. Each year in the United States,pneumococcal diseases account for approximately 50,000 cases ofbacteremia; 3,000 cases of meningitis; 100,000-135,000 hospitalizations;and 7 million cases of otitis media. Pneumococcal infections cause anestimated 40,000 deaths annually in the United States. Children lessthan 2 years of age, adults over 65 years of age and persons of any agewith underlying medical conditions, including, e.g., congestive heartdisease, diabetes, emphysema, liver disease, sickle cell, HIV, and thoseliving in special environments, e.g., nursing homes and long-term carefacilities, at highest risk for infection.

Drug-resistant S. pneumoniae strains have become common in the UnitedStates, with many penicillin-resistant pneumococci also resistant toother antimicrobial drugs, such as erythromycin ortrimethoprim-sulfamethoxazole.

Treponema pallidium is a spirochete which causes syphilis. T. pallidumis exclusively a pathogen which causes syphilis, yaws and non-venerealendemic syphilis or pinta. Treponema pallidum cannot be grown in vitroand does replicate in the absence of mammalian cells. The initialinfection causes an ulcer at the site of infection; however, thebacteria move throughout the body, damaging many organs overtime. In itslate stages, untreated syphilis, although not contagious, can causeserious heart abnormalities, mental disorders, blindness, otherneurologic problems, and death.

Syphilis is usually treated with penicillin, administered by injection.Other antibiotics are available for patients allergic to penicillin, orwho do not respond to the usual doses of penicillin. In all stages ofsyphilis, proper treatment will cure the disease, but in late syphilis,damage already done to body organs cannot be reversed.

Chlamydia trachomatis is the most common bacterial sexually transmitteddisease in the United States and it is estimated that 4 million newcases occur each year. The highest rates of infection are in 15 to 19year olds. Chlamydia is a major cause of non-gonococcal urethritis(NGU), cervicitis, bacterial vaginitis, and pelvic inflammatory disease(PID). Chlamydia infections may have very mild symptoms or no symptomsat all; however, if left untreated Chlamydia infections can lead toserious damage to the reproductive organs, particularly in women.Antibiotics such as azithromycin, erythromycin, ofloxacin, amoxicillinor doxycycline are typically prescribed to treat Chlamydia infection.

Bartonella henselae Cat Scratch Fever (CSF) or cat scratch disease(CSD), is a disease of humans acquired through exposure to cats, causedby a Gram-negative rod originally named Rochalimaea henselae, andcurrently known as Bartonella henselae. Symptoms include fever andswollen lymph nodes and CSF is generally a relatively benign,self-limiting disease in people, however, infection with Bartonellahenselae can produce distinct clinical symptoms in immunocompromisedpeople, including, acute febrile illness with bacteremia, bacillaryangiomatosis, peliosis hepatis, bacillary splenitis, and other chronicdisease manifestations such as AIDS encephalopathy.

The disease is treated with antibiotics, such as doxycycline,erythromycin, rifampin, penicillin, gentamycin, ceftriaxone,ciprofloxacin, and azithromycin.

Haemophilus influenzae (H. influenza) is a family of Gram-negativebacteria; six types of which are known, with most H. influenza-relateddisease caused by type B, or “HIB”. Until a vaccine for HIB wasdeveloped, HIB was a common causes of otitis media, sinus infections,bronchitis, the most common cause of meningitis, and a frequent culpritin cases of pneumonia, septic arthritis (joint infections), cellulitis(infections of soft tissues), and pericarditis (infections of themembrane surrounding the heart). The H. influenza type B bacterium iswidespread in humans and usually lives in the throat and nose withoutcausing illness. Unvaccinated children under age 5 are at risk for HIBdisease. Meningitis and other serious infections caused by H. influenzainfection can lead to brain damage or death.

Shigella dysenteriae (Shigella dys.) is a Gram-negative rod which causesdysentary. In the colon, the bacteria enter mucosal cells and dividewithin mucosal cells, resulting in an extensive inflammatory response.Shigella infection can cause severe diarrhea which may lead todehydration and can be dangerous for the very young, very old orchronically ill. Shigella dys. forms a potent toxin (shiga toxin), whichis cytotoxic, enterotoxic, neurotoxic and acts as a inhibitor of proteinsynthesis. Resistance to antibiotics such as ampicillin and TMP-SMX hasdeveloped, however, treatment with newer, more expensive antibioticssuch as ciprofloxacin, norfloxacin and enoxacin, remains effective.

Listeria is a genus of Gram-positive, motile bacteria found in human andanimal feces. Listeria monocytogenes causes such diseases aslisteriosis, meningoencephalitis and meningitis. This organism is one ofthe leading causes of death from food-borne pathogens especially inpregnant women, newborns, the elderly, and immunocompromisedindividuals. It is found in environments such as decaying vegetablematter, sewage, water, and soil, and it can survive extremes of bothtemperatures and salt concentration making it an extremely dangerousfood-born pathogen, especially on food that is not reheated. Thebacterium can spread from the site of infection in the intestines to thecentral nervous system and the fetal-placental unit. Meningitis,gastroenteritis, and septicemia can result from infection. In cattle andsheep, listeria infection causes encephalitis and spontaneous abortion.

Proteus mirabilis is an enteric, Gram-negative commensal organism,distantly related to E. coli. It normally colonizes the human urethra,but is an opportunistic pathogen that is the leading cause of urinarytract infections in catheterized individuals. P. mirabilis has twoexceptional characteristics: 1) it has very rapid motility, whichmanifests itself as a swarming phenomenon on culture plates; and 2) itproduce urease, which gives it the ability to degrade urea and survivein the genitourinary tract.

Yersinia pestis is the causative agent of plague (bubonic and pulmonary)a devastating disease which has killed millions worldwide. The organismcan be transmitted from rats to humans through the bite of an infectedflea or from human-to-human through the air during widespread infection.Yersinia pestis is an extremely pathogenic organism that requires veryfew numbers in order to cause disease, and is often lethal if leftuntreated. The organism is enteroinvasive, and can survive and propagatein macrophages prior to spreading systemically throughout the host.

Bacillus anthracis is also known as anthrax. Humans become infected whenthey come into contact with a contaminated animal. Anthrax is nottransmitted due to person-to-person contact. The three forms of thedisease reflect the sites of infection which include cutaneous (skin),pulmonary (lung), and intestinal. Pulmonary and intestinal infectionsare often fatal if left untreated. Spores are taken up by macrophagesand become internalized into phagolysozomes (membranous compartment)whereupon germination initiates. Bacteria are released into thebloodstream once the infected macrophage lyses whereupon they rapidlymultiply, spreading throughout the circulatory and lymphatic systems, aprocess that results in septic shock, respiratory distress and organfailure. The spores of this pathogen have been used as a terror weapon.

Burkholderia mallei is a Gram-negative aerobic bacterium that causesGlanders, an infectious disease that occurs primarily in horses, mules,and donkeys. It is rarely associated with human infection and is morecommonly seen in domesticated animals. This organism is similar to B.pseudomallei and is differentiated by being nonmotile. The pathogen ishost-adapted and is not found in the environment outside of its host.Glanders is often fatal if not treated with antibiotics, andtransmission can occur through the air, or more commonly when in contactwith infected animals. Rapid-onset pneumonia, bacteremia (spread of theorganism through the blood), pustules, and death are common outcomesduring infection. The virulence mechanisms are not well understood,although a type III secretion system similar to the one from Salmonellatyphimurium is necessary. No vaccine exists for this potentiallydangerous organism which is thought to have potential as a biologicalterror agent. The genome of this organism carries a large number ofinsertion sequences as compared to the related Bukholderia pseudomallei(below), and a large number of simple sequence repeats that may functionin antigenic variation of cell surface proteins.

Burkholderia pseudomallei is a Gram-negative bacterium that causesmeliodosis in humans and animals. Meliodosis is a disease found incertain parts of Asia, Thailand, and Australia. B. pseudomallei istypically a soil organism and has been recovered from rice paddies andmoist tropical soil, but as an opportunistic pathogen can cause diseasein susceptible individuals such as those that suffer from diabetesmellitus. The organism can exist intracellularly, and causes pneumoniaand bacteremia (spread of the bacterium through the bloodstream). Thelatency period can be extremely long, with infection preceding diseaseby decades, and treatment can take months of antibiotic use, withrelapse a commonly observed phenomenon. Intercellular spread can occurvia induction of actin polymerization at one pole of the cell, allowingmovement through the cytoplasm and from cell-to-cell. This organismcarries a number of small sequence repeats which may promoter antigenicvariation, similar to what was found with the B. mallei genome.

Burkholderia cepacia is a Gram-negative bacterium composed of at leastseven different sub-species, including Burkholderia multivorans,Burkholderia vietnamiensis, Burkholderia stabilis, Burkholderiacenocepacia and Burkholderia ambifaria. B. cepacia is an important humanpathogen which most often causes pneumonia in people with underlyinglung disease (such as cystic fibrosis or immune problems (such as(chronic granulomatous disease). B. cepacia is typically found in waterand soil and can survive for prolonged periods in moist environments.Person-to-person spread has been documented; as a result, manyhospitals, clinics, and camps for patients with cystic fibrosis haveenacted strict isolation precautions B. cepacia. Individuals with thebacteria are often treated in a separate area than those without tolimit spread. This is because infection with B. cepacia can lead to arapid decline in lung function resulting in death. Diagnosis of B.cepacia involves isolation of the bacteria from sputum cultures.Treatment is difficult because B. cepacia is naturally resistant to manycommon antibiotics including aminoglycosides (such as tobramycin) andpolymixin B. Treatment typically includes multiple antibiotics and mayinclude ceftazidime, doxycycline, piperacillin, chloramphenicol, andco-trimoxazole.

Francisella tularensis was first noticed as the causative agent of aplague-like illness that affected squirrels in Tulare County inCalifornia in the early part of the 20th century by Edward Francis. Theorganism now bears his namesake. The disease is called tularemia and hasbeen noted throughout recorded history. The organism can be transmittedfrom infected ticks or deerflies to a human, through infected meat, orvia aerosol, and thus is a potential bioterrorism agent. It is anaquatic organism, and can be found living inside protozoans, similar towhat is observed with Legionella. It has a high infectivity rate, andcan invade phagocytic and nonphagocytic cells, multiplying rapidly. Oncewithin a macrophage, the organism can escape the phagosome and live inthe cytosol.

Veterinary Applications

A healthy microflora in the gastro-intestinal tract of livestock is ofvital importance for health and corresponding production of associatedfood products. As with humans, the gastrointestinal tract of a healthyanimal contains numerous types of bacteria (i.e., E. coli, Pseudomonasaeruginosa and Salmonella spp.), which live in ecological balance withone another. This balance may be disturbed by a change in diet, stress,or in response to antibiotic or other therapeutic treatment, resultingin bacterial diseases in the animals generally caused by bacteria suchas Salmonella, Campylobacter, Enterococci, Tularemia and E. coli.Bacterial infection in these animals often necessitates therapeuticintervention, which has treatment costs as well being frequentlyassociated with a decrease in productivity.

As a result, livestock are routinely treated with antibiotics tomaintain the balance of flora in the gastrointestinal tract. Thedisadvantages of this approach are the development of antibioticresistant bacteria and the carry over of such antibiotics and theresistant bacteria into resulting food products for human consumption.

C2. Target Sequences for Cell-Division and Cell-Cycle Target Proteins

The antisense oligomers of the claimed subject matter are designed tohybridize to a region of a bacterial mRNA that encodes an essentialbacterial gene. Exemplary genes are those required for cell division,cell cycle proteins, or genes required for lipid biosynthesis or nucleicacid replication. Any essential bacterial gene can be targeted once agene's essentiality is determined. One approach to determining whichgenes in an organism are essential is to use genetic footprintingtechniques as described (Gerdes, Scholle et al. 2003). In this report,620 E. coli genes were identified as essential and 3,126 genes asdispensable for growth under culture conditions for robust aerobicgrowth. Evolutionary context analysis demonstrated that a significantnumber of essential E. coli genes are preserved throughout the bacterialkingdom, especially the subset of genes for key cellular processes suchas DNA replication, cell division and protein synthesis.

In various aspects, the claimed subject matter provides an antisenseoligomer which is a nucleic acid sequence effective to stably andspecifically bind to a nucleic acid target sequence which encodes anessential bacterial protein including the following: (1) a sequencespecific to a particular strain of a given species of bacteria, such asa strain of E. coli associated with food poisoning, e.g., O157:H7 (seeTable 1 below); (2) a sequence common to two or more species ofbacteria; (3) a sequence common to two related genera of bacteria (i.e.,bacterial genera of similar phylogenetic origin); (4) a sequencegenerally conserved among Gram-negative bacteria; (5) generallyconserved among Gram-positive bacteria; or (6) a consensus sequence foressential bacterial protein-encoding nucleic acid sequences in general.

In general, the target for modulation of gene expression using theantisense methods of the claimed subject matter comprises an mRNAexpressed during active bacterial growth or replication, such as an mRNAsequence transcribed from a gene of the cell division and cell wallsynthesis (dcw) gene cluster, including, but not limited to, zipA, sulA,secA, dicA, dicB, dicC, dicF, ftsA, ftsI, ftsN, ftsK, ftsL, ftsQ, ftsW,ftsZ, murC, murD, murE, murF, murG, minC, minD, minE, mraY, mraW, mraZ,seqA and dd/B. See (Bramhill 1997), and (Donachie 1993), both of whichare expressly incorporated by reference herein, for general reviews ofbacterial cell division and the cell cycle of E. coli, respectively.Additional targets include genes involved in lipid biosynthesis (e.g.acpP) and replication (e.g. gyrA).

Cell division in E. coli involves coordinated invagination of all 3layers of the cell envelope (cytoplasmic membrane, rigid peptidoglycanlayer and outer membrane). Constriction of the septum severs the cellinto 2 compartments and segregates the replicated DNA. At least 9essential gene products participate in this process: ftsZ, ftsA, ftsQ,ftsL, ftsI, ftsN, ftsK, ftsW and zipA (Hale and de Boer 1999). Preferredprotein targets are the three discussed below, and in particular, theGyrA and AcpP targets described below.

FtsZ, one of the earliest essential cell division genes in E. coli, is asoluble, tubulin-like GTPase that forms a membrane-associated ring atthe division site of bacterial cells. The ring is thought to drive cellconstriction, and appears to affect cell wall invagination. FtsZ bindsdirectly to a novel integral inner membrane protein in E. coli calledzipA, an essential component of the septal ring structure that mediatescell division in E. coli (Lutkenhaus and Addinall 1997).

GyrA refers to subunit A of the bacterial gyrase enzyme, and the genetherefore. Bacterial gyrase is one of the bacterial DNA topoisomerasesthat control the level of supercoiling of DNA in cells and is requiredfor DNA replication.

AcpP encodes acyl carrier protein, an essential cofactor in lipidbiosynthesis. The fatty acid biosynthetic pathway requires that the heatstable cofactor acyl carrier protein binds intermediates in the pathway.

For each of these three proteins, Table 1 below provides exemplarybacterial sequences which contain a target sequence for each of a numberof important pathogenic bacteria. The gene sequences are derived fromthe GenBank Reference full genome sequence for each bacterial strain(http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi). The gene location oneither the positive (+) or negative (−) strand of the genome is listedunder “Strand”, it being recognized that the strand indicated is thecoding sequence for the protein, that is, the sequence corresponding tothe mRNA target sequence for that gene. For example, the two E. coligenes (ftsZ and acpP) in which the coding sequence is on the positivestrand, the sequence is read 5′ to 3′ in the left-to-right direction.Similarly for the E. coli gyrA gene having the coding region on theminus genomic strand, the coding sequence is read as the reversecomplement in the right to left direction (5′ to 3′).

Based on these considerations, exemplary targeting sequences for use inpracticing the claimed invention are those having between 10-25,preferably 10-20 bases, preferably complete but at least 10-basecomplementarity with the mRNA target sequence, and complementary to aregion of the mRNA that includes the AUG start site or a region up to 20bases downstream of the start site. Where the compound is used ininhibiting infection by one of the bacteria identified in the tablebelow, by inhibiting one of the three identified bacterial proteins, theantisense oligomer compound has a sequence that is complementary to atleast 10 contiguous bases of the corresponding target sequence indicatedin the table, where these target sequences are identified in thesequence listing below by SEQ ID NOS:1-61.

TABLE 1 Exemplary bacterial target regions Organism Target SEQ ID(GenBank Ref.) Gene Nucleotide Region NO. Escherichia coil ftsZ105295-105325 1 (NC 000913) acpP 1150828-1150858 2 gyrA 2337422-23374523 Escherichia coli 0157:H7 ftsZ 109901-109931 4 (NC 002655) acpP1595786-1595816 5 gyrA 3136439-3136469 6 Salmonella thyphimurium ftsZ155673-155703 7 (NC 003197) acpP 1280103-1280133 8 gyrA 2376327-23763579 Pseudomonas aeruginosa ftsZ 4940463-4940493 10 (NC 002516) acpP3325162-3325192 11 gyrA 3559177-3559207 12 Vibrio cholera ftsZ2566223-2566253 13 (NC 002505) acpP 254495-254525 14 gyrA1330197-1330227 15 Neisseria gonorrhoea ftsZ 1500031-1500060 16 (NC002946) acpP 1724391-1724420 17 gyrA 621170-621199 18 Staphylococcusaureus ftsZ 1165772-1165802 19 (NC 002745) gyrA 6995-7025 20 fmhB2322402-2322431 21 Mycobacterium tuberculosis ftsZ 2408265-2408295 22(NC 002755) acpP 1510172-1510202 23 gyrA 7292-7322 24 pimA2935126-2935126 25 cysS2 4015924-4015953 26 Helicobacter pylori ftsZ1042227-1042257 27 (NC 000915) acpP 594253-594283 28 gyrA 752502-75253229 Streptococcus pneumoniae ftsZ 1566686-1566716 30 (NC 003028) acpP396681-396711 31 gyrA 1149835-1149865 32 Treponema palladium ftsZ414741-414771 33 (NC 000919) acpP 877626-877656 34 gyrA 4381-4411 35Chlamydia trachomatis acpP 263915-263945 36 (NC 000117) gyrA756474-756504 37 Bartonella henselae ftsZ 1232075-1232104 38 (NC 005956)acpP 623133-623162 39 gyrA 1123338-1123367 40 Hemophilis influenza ftsZ1212011-1212041 41 (NC 000907) acpP 171140-171170 42 gyrA1344341-1344371 43 Listeria monocytogenes ftsZ 2102288-2102307 44 (NC002973) acpP 1860519-1860548 45 gyrA 8055-8084 46 Yersinia pestis ftsZ605864-605893 47 (NC 003143) acpP 1824110-1824139 48 gyrA1370719-1370748 49 Bacillus anthracis ftsZ 3725338-3725367 50 (NC005945) acpP 3666877-3666906 51 gyrA 6586-6615 52 Burkholderia malleiftsZ 2650793-2650822 53 (NC 006348) acpP 559420-559449 54 gyrA461883-461912 55 Burkholderia pseudomallei ftsZ 3600339-3600368 56 (NC006350) acpP 2945187-2945216 57 gyrA 3039114-3039143 58 Francisellatularensis ftsZ 203738-203767 59 (NC 006570) acpP 1421890-1421919 60gyrA 1639887-1639916 61

Any essential bacterial gene can be targeted using the methods of theclaimed subject matter. As described above, an essential bacterial genefor any bacterial species can be determined using a variety of methodsincluding those described by Gerdes for E. coli (Gerdes, Scholle et al.2003). Many essential genes are conserved across the bacterial kingdomthereby providing additional guidance in target selection. Targetregions can be obtained using readily available bioinformatics resourcessuch as those maintained by the National Center for BiotechnologyInformation (NCBI). Complete reference genomic sequences for a largenumber of microbial species can be obtained (e.g., seehttp://www.ncbi.nlm.nih.gov/genomes/lproks.cgi) and sequences foressential bacterial genes identified. Bacterial strains can be obtainedfrom the American Type Culture Collection (ATCC). Simple cell culturemethods, such as those described in the Examples, using the appropriateculture medium and conditions for any given species, can be establishedto determine the antibacterial activity of antisense compounds. Once asuitable targeting antisense oligomer has been identified, the peptidemoieties of the compounds can be altered to obtain optimal antibacterialactivity. An optimal peptide moiety can then be fixed and alternativeantisense moieties tested for improved antibacterial activity. One ormore iterations of this process can lead to compounds with improvedactivity but, in general, no more than two iterations are needed toidentify highly active antibacterial agents.

Thus, the first step in selecting a suitable antisense compound is toidentify, by the methods above, a targeting sequence that includes theAUG start site and/or contains at least about 10-20 bases downstream ofthe start site. For purposes of illustration, assume that the antisensecompound to be prepared is for use in inhibiting an E. coli bacterialinfection in an individual infected with E. coli strain 0157:H7, andthat the essential gene being targeted is the E. coli acpP gene. Onesuitable target sequence for this gene identified by the methods aboveis SEQ ID NO:2 having the sequence 5′-ATTTAAGAGTATGAGCACTATCGAAGAACGC-3′where the sequence gives the DNA thymine (T) bases rather than the RNAuracil (U) bases, and where the AUG start site (ATG) is shown in bold.

Again, for purposes of illustration, four model antisense targetingsequences, each of them 11 bases in length, are selected: (i) anantisense sequence that spans the AUG start site with four bases of eachside and has the sequence identified by SEQ ID NO:94; (ii) an antisensesequence that overlaps the AUG starts at its 5′ end and extends in a 3′direction an additional 8 bases into the coding region of the gene,identified as SEQ ID NO:95; (iii) an antisense sequence complementary tobases 5-15 of the gene's coding region, identified as SEQ ID NO:66, and(iv) an antisense sequence complementary to bases 11-21 of the gene'scoding region, identified as SEQ ID NO:96.

Once antisense sequences have been selected and the antisense compoundsynthesized and conjugated to arginine and/or lysine-containingpeptides, the compounds may be tested for the ability to inhibitbacterial growth, in this case growth of an E. coli strain in culture.Following the protocol in Example 1, for example, the four 11 mersequences described above are individually tested for optimal activity,e.g., maximum drop in CFU/ml at a given dose, e.g., 5-20 DM, against anE. coli culture. Compound(s) showing optimal activity are then tested inanimal models, as described in Example 2, or veterinary animals, priorto use for treating human infection.

C2. Target Sequences for Bacterial 16S Ribosomal RNA

In one embodiment, the antisense oligomers of the invention are designedto hybridize to a region of a bacterial 16S rRNA nucleic acid sequenceunder physiological conditions, with a T_(m) substantially greater than37° C., e.g., at least 45° C. and preferably 60° C.-80° C. The oligomeris designed to have high binding affinity to the nucleic acid and may be100% complementary to the 16S rRNA nucleic acid target sequence, or itmay include mismatches, as further described above.

More particularly, the antisense oligonucleotide that is enhanced inactivity, in accordance with the invention, has a targeting sequencethat is effective to stably and specifically bind to a target 16S rRNAsequences which has one or more of the following characteristics: (1) asequence found in a double stranded region of a 16s rRNA, e.g., thepeptidyl transferase center, the alpha-sarcin loop and the mRNA bindingregion of the 16S rRNA sequence; (2) a sequence found in a singlestranded region of a bacterial 16s rRNA; (3) a sequence specific to aparticular strain of a given species of bacteria, i.e., a strain of E.coli associated with food poisoning; (4) a sequence specific to aparticular species of bacteria; (5) a sequence common to two or morespecies of bacteria; (6) a sequence common to two related genera ofbacteria (i.e., bacterial genera of similar phylogenetic origin); (7) asequence generally conserved among Gram-negative bacterial 16S rRNAsequences; (6) a sequence generally conserved among Gram-positivebacterial 16S rRNA sequences; or (7) a consensus sequence for bacterial16S rRNA sequences in general.

Exemplary bacteria and associated GenBank Accession Nos. for 16S rRNAsequences are provided in Table 1 of above-referenced U.S. Pat. No.6,677,153.

It will be understood that one of skill in the art may readily determineappropriate targets for antisense oligomers, and design and synthesizeantisense oligomers using techniques known in the art. Targets can beidentified by obtaining the sequence of a target 16S or 23 S nucleicacid of interest (e.g. from GenBank) and aligning it with other 16S or23S nucleic acid sequences using, for example, the MacVector 6.0program, a ClustalW algorithm, the BLOSUM 30 matrix, and defaultparameters, which include an open gap penalty of 10 and an extended gappenalty of 5.0 for nucleic acid alignments. An alignment may also becarried out using the Lasergene99 MegAlign Multiple Alignment programwith a ClustalW algorithm run under default parameters.

For example, given the 16s rRNA sequences provided in Table 1 and other16s rRNA sequences available in GenBank, one of skill in the art canreadily align the 16s rRNA sequences of interest and determine whichsequences are conserved among one or more different bacteria, and thosewhich are specific to one or more particular bacteria. A similaralignment can be performed on 23 S rRNA sequences.

As an illustration, the 16S rRNA sequences from the organisms shown inTable 1 were aligned using the Lasergene 99 MegAlign Multiple Alignmentprogram, with a ClustalW algorithm and default parameters. Tables 2-5 ofthe above-referenced U.S. Pat. No. 6,677,153. show exemplary oligomersantisense to 16S rRNA of these bacterial species, including sequencestargeting individual bacteria, multiple bacteria, and broad spectrumsequences. These oligomers were derived from the sequences in Table 1and from the alignment performed as described above. As the Tables show,a number of sequences were conserved among different organisms.

III. Antibacterial Activity in Cell Culture

Antisense oligonucleotide compounds having enhanced activity by virtueof having an arginine-rich carrier peptide and/or a positively chargedbackbone, and directed against the AUG start site region of thebacterial AcpP gene were tested for their ability to inhibit bacterialgrowth in culture. These studies are reported in Example 21, withreference to FIGS. 3A and 3B. As seen in the latter figure, a strikinginhibition was observed for antisense compounds having between 10 and 14bases, with nearly complete inhibition being observed for the compoundwith an 11-base length. As with the expression studies involving markergenes described above, the results for inhibition of a bacterial gene inbacteria are unpredictable from the behavior of the same antisensecompounds in a cell-free bacterial system. As seen in FIG. 4, strongestinhibition was observed for antisense compounds between 11 and 20 bases.

As described in Example 23 and shown in FIGS. 7 to 16, conjugation of anarginine-rich peptide to the antisense oligonucleotides described abovegreatly enhance their antibacterial properties. Exemplary amino acidsequences and the peptides used in experiments in support of the claimedsubject matter are listed in Table 4 below as SEQ ID NOS:79-93. Oneexemplary peptide is named RFF (SEQ ID NO:79) and consists of thesequence N-RFFRFFRFFAhxβAla-COOH (using the standard one letter aminoacid code and Ahx for 6-aminohexanoic acid and βAla for beta-alanine),and has three repeating Arg-Phe-Phe residues. This peptide isrepresentative of one model peptide having at least two, preferablythree repeating Arg-Phe-Phe units. One exemplary peptide-conjugated PMO(P-PMO) derived from the RFF peptide (RFF-AcpP11, SEQ ID NO:79)demonstrated the ability to reduce the CFU/ml of E. coli strains by asmuch as five orders of magnitude (FIG. 11) and an IC₅₀ of 3.7 μM that islower than the 50% inhibitory concentration (IC₅₀) observed forampicillin (7.7 μM) under the same culture conditions (FIG. 14).

Another exemplary peptide named (RAhxR)₄ consisting of the sequenceRAhxRRAhxRRAhxRRAhxRAhxβAla-COOH (SEQ ID NO:97) contains 4 repeatingArg-Ahx-Arg sequences. An exemplary conjugate derived from the (RAhxR)₄(SEQ ID NO: 112) peptide ([RAhR]₄-AcpP+, SEQ ID NO: 98) demonstrated aminimum inhibitory concentration (MIC) of 0.313 μM, a 32-fold reductionin the MIC versus ampicillin (10.0 μM), as measured against E. coliW3110 in the broth microdilution method of the Clinical and LaboratoryStandards Institute (FIG. 2O).

The carrier peptides have the ability, when conjugated to the 5′- or3′-end of the anti-bacterial antisense PMO compound having SEQ ID NO:66,to enhance the anti-bacterial activity of the PMO compound by a factorof at least 10 and typically at least 10², as measured by the reductionin bacterial colony-forming units/mi (CFU/ml) when thepeptide-conjugated PMO (P-PMO) compound is added at a concentration of20 μM in a culture to E. coli, strain W3110 at 5×10⁷ CFU/ml for a periodof 8 hours at 37° C. with aeration, relative to the activity of the PMOcompound alone. The insertion of cationic linkages, as shown in FIG. 2H,into the P-PMO backbone to form a P-PMO+ enhances the antibacterialactivity of the conjugate by a factor of 3-fold, as measured in vivo bythe increase in survival and the reduction in bacterial CFU/mL, relativeto the activity of the uncharged P-PMO compound alone.

Thus, the carrier peptides and/or charged backbone linkages describedabove are capable of enhancing the activity of a morpholino antisenseoligonucleotide up to 100-1,000 times or more. It will be appreciatedthat selection of additional carrier peptide sequences within thegeneral description of the carrier peptide above, and for optimizing thenumber and distribution of charged groups along the oligonucleotidebackbone can be carried out with the above cell-culture test methods.For example, the oligonucleotide conjugate with charged backbone groupsis added, at a concentration of 20 μM in a culture to E. coli, strainW3110 for a period of 8 hours, with the oligonucletide alone (control)being added at a similar concentration to a culture of the samebacteria. After an eight-hour incubation time, the number of colonyforming units per ml (CFU/ml) are measured in both the “conjugate” or“conjugate-charged-backbone” or “charged-backbone” and control cultures.If the CFU/ml count for the enhanced oligonucleotide culture is morethan 100-fold less, and preferably a 1000-fold less than that of thecontrol culture, the peptide can be identified as one suitable for useas a high-activity antibacterial agent.

IV. Method for Inhibiting Bacteria

The enhanced-activity oligonucleotide of the invention is useful in amethod of inhibiting bacterial infection, by exposing the infectingbacteria to the enhanced-activity compound, as illustrated by the studyreported in Example 21 and described above with respect to FIGS. 3A and3B. The activities reporter here were determined with a representative“PMO,” “P-PMO,” or “P-PMO+” oligonucleotide as representative of anoligonucleotide with phosphorus-containing backbone linkages.

In one aspect, the method is applied to inhibiting a bacterial infectionin a mammalian subject, including a human subject, by administering theantisense compound to the subject in a therapeutic amount. Todemonstrate the method, groups of 12 mice were injected IP with E. coliAS19, which has a genetic defect that makes it abnormally permeable tohigh MW solutes. Immediately following infection, each mouse wasinjected IP with 300 μg of an 11-base PMO complementary to acpP (SEQ IDNO:66), an 11-base nonsense sequence PMO, or PBS, as detailed in Example22. As seen in FIG. 5, mice treated with the target antisense showed areduction in bacterial CFUs of about 600 at 23 hours, compared withcontrol treatment.

The same PMOs were again tested, except with E. coli SM105, which has anormal outer membrane. In this method, acpP PMO reduced CFU by 84%compared to nonsense PMO at 12 hours post-infection. There was noreduction of CFU at 2, 6, or 24 hours (FIG. 6). Mice were injected witha second dose at 24 hours post-infection. By 48 hours post-infection theCFU of acpP PMO-treated mice were 70% lower than the CFU of nonsensePMO-treated mice (FIG. 6).

To demonstrate that the effect on bacterial infection was sequencespecific, a luciferase reporter gene whose expression would not affectgrowth was used, and luciferase expression was measured directly by twoindependent criteria, luciferase activity and luciferase proteinabundance. As detailed in Example 2, the study demonstrated that anantisense compound complementary to the luciferase mRNA inhibitedluciferase expression at two different times after administration of thePMO. Moreover, inhibition was quantitatively similar with both methodsof measurement. These results show directly that PMO inhibit bacterialtarget gene expression in vivo in a sequence-specific manner.

An improved antibacterial PMO can be obtained by conjugating a short6-14 amino acid peptide that enhances either intracellular delivery orantisense activity or both. Exemplary peptides and peptide-conjugatedPMO (P-PMO) are listed in Table 4 as SEQ ID NOS:88-93. As described inExample 23, enhanced antibacterial activity was observed in pure cultureexperiments using a variety of E. coli and S. typhimurium strainsincluding the clinically isolated enteropathogenic E. coli (EPEC) strain0127:H6. Example 24 describes the antibacterial activity ofpeptide-conjugated PMO targeted to B. cenocepacia and P. aeruginosa.

In one application of the enhanced-activity compounds, the method isapplied to inhibiting a bacterial infection in a mammalian subject,including a human subject, by administering the antisense compound tothe subject in a therapeutic amount. To demonstrate the method, groupsof 2 to 4 mice were injected IP with E. coli W3110. Immediatelyfollowing infection and again 12 hours later, each mouse was injected IPwith water control or 1, 10, 30, 100, or 300 μg of an 11-base P-PMO+complementary to acpP (SEQ ID NO:98), as detailed in Example 5. As seenin FIGS. 19A-C, mice treated with 10 tg and above of theP-PMO+(oligonucleotide with conjugate carrier peptide and chargedbackbone linkages) showed full protection up to termination of the studyat 48 hours after infection, and a dose-dependent reduction in bacterialCFUs ranging from 0.5 to 4 magnitudes, compared with control treatment.

It will be understood that the in vivo efficacy of such a P-PMO+ in asubject using the methods of the claimed invention is dependent uponnumerous factors including, but not limited to, (1) the target sequence;(2) the duration, dose and frequency of antisense administration; and(3) the general condition of the subject.

In other cases, the antisense oligonucleotides of the claimed subjectmatter find utility in the preparation of anti-bacterial vaccines. Inthis aspect of the claimed subject matter, a culture of a particulartype of bacteria is incubated in the presence of a P-PMO+ of the typedescribed above, in an amount effective to produce replication-crippledand/or morphologically abnormal bacterial cells. Suchreplication-crippled and/or morphologically abnormal bacterial cells areadministered to a subject and act as a vaccine.

The efficacy of an in vivo administered antisense oligomer of theclaimed subject matter in inhibiting or eliminating the growth of one ormore types of bacteria may be determined by in vitro culture ormicroscopic examination of a biological sample (tissue, blood, etc.)taken from a subject prior to, during and subsequent to administrationof the P-PMO+. (See, for example, (Pari, Field et al. 1995); and(Anderson, Fox et al. 1996). The efficacy of an in vivo administeredvaccine of P-PMO+-treated bacteria may be determined by standardimmunological techniques for detection of an immune response, e.g.,ELISA, Western blot, radioimmunoassay (RIA), mixed lymphocyte reaction(MLR), assay for bacteria-specific cytotoxic T lymphocytes (CTL), etc.

A. Administration Methods

Effective delivery of the P-PMO+ to the target nucleic acid is animportant aspect of treatment. In accordance with the claimed invention,such routes of delivery include, but are not limited to, varioussystemic routes, including oral and parenteral routes, e.g.,intravenous, subcutaneous, intraperitoneal, and intramuscular, as wellas inhalation, transdermal and topical delivery. The appropriate routemay be determined by one of skill in the art, as appropriate to thecondition of the subject under treatment. For example, an appropriateroute for delivery of a P-PMO+ in the treatment of a bacterial infectionof the skin is topical delivery; while delivery of a P-PMO+ in thetreatment of a bacterial respiratory infection is by inhalation. Methodseffective to deliver the oligomer to the site of bacterial infection orto introduce the compound into the bloodstream are also contemplated.

Transdermal delivery of P-PMO+ may be accomplished by use of apharmaceutically acceptable carrier adapted for e.g., topicaladministration. One example of morpholino oligomer delivery is describedin PCT patent application WO 97/40854, incorporated herein by reference.

In one preferred embodiment, the compound is a P-PMO+, contained in apharmaceutically acceptable carrier, and is delivered orally.

The P-PMO+ may be administered in any convenient vehicle which isphysiologically acceptable. Such a composition may include any of avariety of standard pharmaceutically accepted carriers employed by thoseof ordinary skill in the art. Examples of such pharmaceutical carriersinclude, but are not limited to, saline, phosphate buffered saline(PBS), water, aqueous ethanol, emulsions such as oil/water emulsions,triglyceride emulsions, wetting agents, tablets and capsules. It will beunderstood that the choice of suitable physiologically acceptablecarrier will vary dependent upon the chosen mode of administration.

In some instances liposomes may be employed to facilitate uptake of theantisense oligonucleotide into cells. (See, e.g., Williams, S. A.,Leukemia 10(12):1980-1989, 1996; Lappalainen et al., Antiviral Res.23:119, 1994; Uhlmann et al., ANTISENSE OLIGONUCLEOTIDES: A NEWTHERAPEUTIC PRINCIPLES , Chemical Reviews, Volume 90, No. 4, pages544-584, 1990; Gregoriadis, G., Chapter 14, Liposomes, Drug Carriers inBiology and Medicine, pp. 287-341, Academic Press, 1979). Hydrogels mayalso be used as vehicles for antisense oligomer administration, forexample, as described in WO 93/01286. Alternatively, theoligonucleotides may be administered in microspheres or microparticles.(See, e.g., Wu, G. Y. and Wu, C. H., J. Biol. Chem. 262:4429-4432,1987.)

Sustained release compositions are also contemplated within the scope ofthis application. These may include semipermeable polymeric matrices inthe form of shaped articles such as films or microcapsules.

Typically, one or more doses of P-PMO+ are administered, generally atregular intervals for a period of about one to two weeks. Preferreddoses for oral administration are from about 10 mg oligomer/patient toabout 250 mg oligomer/patient (based on a weight of 70 kg). In somecases, doses of greater than 250 mg oligomer/patient may be necessary.For IV administration, the preferred doses are from about 1.0 mgoligomer/patient to about 100 mg oligomer/patient (based on an adultweight of 70 kg). The P-PMO+ is generally administered in an amount andmanner effective to result in a peak blood concentration of at least200-400 nM oligomer.

In a further aspect of this embodiment, a P-PMO+ is administered atregular intervals for a short time period, e.g., daily for two weeks orless. However, in some cases the P-PMO+ is administered intermittentlyover a longer period of time. Administration of a P-PMO+ to a subjectmay also be followed by, or concurrent with, administration of anantibiotic or other therapeutic treatment.

In one aspect of the method, the subject is a human subject, e.g., apatient diagnosed as having a localized or systemic bacterial infection.The condition of a patient may also dictate prophylactic administrationof a P-PMO+ of the claimed subject matter or a P-PMO+-treated bacterialvaccine, e.g. in the case of a patient who (1) is immunocompromised; (2)is a burn victim; (3) has an indwelling catheter; or (4) is about toundergo or has recently undergone surgery.

In another application of the method, the subject is a livestock animal,e.g., a chicken, turkey, pig, cow or goat, etc, and the treatment iseither prophylactic or therapeutic. The invention also includes alivestock and poultry food composition containing a food grainsupplemented with a subtherapeutic amount of an antibacterial antisensecompound of the type described above. Also contemplated is in a methodof feeding livestock and poultry with a food grain supplemented withsubtherapeutic levels of an antibiotic, an improvement in which the foodgrain is supplemented with a subtherapeutic amount of an antibacterialoligonucleotide composition as described above.

The methods of the invention are applicable, in general, to treatment ofany condition wherein inhibiting or eliminating the growth of bacteriawould be effective to result in an improved therapeutic outcome for thesubject under treatment.

One aspect of the invention is a method for treatment of a bacterialinfection which includes the administration of a morpholino antisenseoligomer to a subject, followed by or concurrent with administration ofan antibiotic or other therapeutic treatment to the subject.

B. Treatment Monitoring Methods

It will be understood that an effective in vivo treatment regimen usingthe P-PMO+ compounds of the invention will vary according to thefrequency and route of administration, as well as the condition of thesubject under treatment (i.e., prophylactic administration versusadministration in response to localized or systemic infection).Accordingly, such in vivo therapy will generally require monitoring bytests appropriate to the particular type of bacterial infection undertreatment and a corresponding adjustment in the dose or treatmentregimen in order to achieve an optimal therapeutic outcome.

The efficacy of a given therapeutic regimen involving the methodsdescribed herein may be monitored, e.g., by general indicators ofinfection, such as complete blood count (CBC), nucleic acid detectionmethods, immunodiagnostic tests, or bacterial culture.

Identification and monitoring of bacterial infection generally involvesone or more of (1) nucleic acid detection methods, (2) serologicaldetection methods, i.e., conventional immunoassay, (3) culture methods,and (4) biochemical methods. Such methods may be qualitative orquantitative.

Nucleic acid probes may be designed based on publicly availablebacterial nucleic acid sequences, and used to detect target genes ormetabolites (i.e., toxins) indicative of bacterial infection, which maybe specific to a particular bacterial type, e.g., a particular speciesor strain, or common to more than one species or type of bacteria (i.e.,Gram positive or Gram negative bacteria). Nucleic amplification tests(e.g., PCR) may also be used in such detection methods.

Serological identification may be accomplished using a bacterial sampleor culture isolated from a biological specimen, e.g., stool, urine,cerebrospinal fluid, blood, etc. Immunoassay for the detection ofbacteria is generally carried out by methods routinely employed by thoseof skill in the art, e.g., ELISA or Western blot. In addition,monoclonal antibodies specific to particular bacterial strains orspecies are often commercially available.

Culture methods may be used to isolate and identify particular types ofbacteria, by employing techniques including, but not limited to, aerobicversus anaerobic culture, growth and morphology under various cultureconditions. Exemplary biochemical tests include Gram stain (Gram, 1884;Gram positive bacteria stain dark blue, and Gram negative stain red),enzymatic analyses (i.e., oxidase, catalase positive for Pseudomonasaeruginosa), and phage typing.

It will be understood that the exact nature of such diagnostic, andquantitative tests as well as other physiological factors indicative ofbacterial infection will vary dependent upon the bacterial target, thecondition being treated and whether the treatment is prophylactic ortherapeutic.

In cases where the subject has been diagnosed as having a particulartype of bacterial infection, the status of the bacterial infection isalso monitored using diagnostic techniques typically used by those ofskill in the art to monitor the particular type of bacterial infectionunder treatment.

The P-PMO+ treatment regimen may be adjusted (dose, frequency, route, asindicated, based on the results of immunoassays, other biochemical testsand physiological examination of the subject under treatment.

From the foregoing, it will be appreciated how various objects andfeatures of the invention are met. The method provides an improvement intherapy against bacterial infection, using P-PMO+ sequences to achieveenhanced cell uptake and anti-bacterial action. As a result, drugtherapy is more effective and less expensive, both in terms of cost andamount of compound required.

An important advantage of the invention is that compounds effectiveagainst virtually any pathogenic bacterium can be readily designed andtested, e.g., for rapid response against new drug-resistant bacteria, orin cases of bioterrorism. Once a target bacterium is identified, thesequence selection methods described allow one to readily identify oneor more likely gene targets, among a number of essential genes, andprepare antisense compounds directed against the identified target.Because clinical testing on the safety and efficacy, once establishedfor a small group of compounds, can be extrapolated to virtually any newtarget, relatively little time is needed in addressing newbacterial-infection challenges as they arise.

The following examples are intended to illustrate but not to limit theinvention.

Materials and Methods

Unless otherwise noted, all chemicals were obtained fromSigma-Aldrich-Fluka. Benzoyl adenosine, benzoyl cytidine, andphenylacetyl guanosine were obtained from Carbosynth Limited, UK

Example 1 Morpholino Subunits (See FIG. 2B)

General Preparation of morpholino salts 3a-d,f: To a cooled mixture ofmethanol (5-10 mL/g ribonucleosides 2) was added a warm aqueous solutionof sodium meta-periodate (1.05 eq). At this stage, the composition ofthe reaction mixture will be from 15-40% water/methanol (v:v). To thismixture was added, in portions, solid 1a-d,f. Upon reaction completion(1-2 hr), the by-product sodium iodate cake was removed by filtrationand reslurried with water/methanol to recover any product intermediate.To the pooled filtrates were added ammonium biborate (14-2.0 eq). Afterstirring at 20° C. for 45-120 min, the mixture was cooled, andborane-triethylamine (1.5-2.0 eq) was added. This mixture was adjustedto pH 3.5-4.0 with a methanolic solution of either p-toluenesulfonicacid [3b, c, d, f] or hydrochloric acid [3a] (4-5 eq). The mixture washeld at pH 3.5-4.0 for 7-14 hr at <10° C. The p-toluenesulfonic acidsalts of 3b, c, d, f were isolated by filtration and purified byrecrystallization/reslurry.

The mixture containing 3a was neutralized to pH 7. The solution wasconcentrated by distillation to remove methanol, and the product wasextracted into 1-butanol. This solution was adjusted to pH 4 with amethanolic solution of oxalic acid (0.5 eq). The oxalic acid salt of 3awas isolated by filtration and purified by reslurry. Yields for3a-d,f=30-75%.

General Preparation of 1a-d,f: Compound 3a-d,f was dissolved/suspendedin N,N-dimethylformamide (4-6 mL/g 3). To this mixture was addedtriethylamine (2.7-3.5 eq) and triphenylmethyl (trityl) chloride(1.1-1.5 eq). Upon reaction completion, the excess trityl chloride wasquenched with diethylamine (0.5 eq). The crude products were isolated byeither direct precipitation from ethyl acetate and water or through anextractive workup (water then ethyl acetate or dichloromethane) andprecipitation. The products were purified by crystallization fromtoluene. Yields=75-90%

Preparation of 1e: Compound if was suspended in dichloromethane (8 mL/g1f). To this suspension were added imidazole (1.3 eq) andt-butyldimethylchlorosilane (1.2 eq). Upon reaction completion (1-2 hr),the solution was washed successively with pH 3 citrate buffer and water.The resulting solution was concentrated to give a foam, which wasdissolved in tetrahydrofuran (8 mL/g if). To this solution were addedpotassium carbonate (2.0 eq) and chloromethyl pivalate (1.5 eq) and themixture was heated to reflux. Upon reaction completion (16 hr), themixture was cooled and diluted with dichloromethane. The mixture waswashed successively with KH₂PO₄ solution (pH 4.5) and water. Theresulting solution was concentrated to give a foam. The foam wasdissolved in tetrahydrofuran (4 mL/g if) and triethylaminetrihydrofluoride (2.0 eq) was added. Upon reaction completion (16 hr),the solution was washed successively with saturated aqueous NaHCO₃ andwater. The product was isolated by solvent exchange into toluene andprecipitation into heptane. Yield=80% of 1e.

Example 2 Morpholino Subunits with Linkage Type (A) (See FIG. 2C)

General Preparation of 5a-e: Compound 1a-e was dissolved indichloromethane (6 mL/g 1) and cooled to <5° C. To this solution wereadded 2,6-lutidine (1.6 eq), N-methylimidazole (0.3 eq), andN,N-dimethylphosphoramidodichloridate 4 (1.6 eq). Upon reactioncompletion (6-12 hr), this mixture was washed with a pH 3 citratebuffer. The crude product was isolated by precipitation into heptane.The final product was purified by silica gel chromatography (gradient ofethyl acetate/heptane). The pooled fractions containing product werecombined, evaporated to a smaller volume, and isolated by precipitationfrom heptane. Yields=40-60%. During the chromatography of subunit 5e, aswell as other subunits derived from this heterocyclic base, followingethyl acetate/heptane elution of the non-polar impurities, a gradient of(5% isopropanol/ethyl acetate) in dichloromethane was used to elute theproduct.

Example 3 Morpholino Subunits with Pro-Cationic Linkages Type (B1) (SeeFIG. 2D)

Preparation of N-trityl piperazine, succinate salt (11): To a cooledsolution of piperazine (10 eq) in toluene/methanol (5:1 toluene/methanol(v:v); 5 mL/g piperazine) was added slowly a solution of trityl chloride10 (1.0 eq) in toluene (5 mL/g trityl chloride). Upon reactioncompletion (1-2 hr), this solution was washed four times with water. Tothe resulting organic solution was added an aqueous solution of succinicacid (1.1 eq; 13 mL water/g succinic acid). This mixture was stirred for90 min, and the solid product was collected by filtration. The crudesolid was purified by two reslurries in acetone. Yield=70%.

Preparation of 1-trifluoroacetyl-4-trityl piperazine (14): To a slurryof 3.0 kg 11 in 18 L methanol (6 mL/g 11) was added 3.51 Ldiisopropylethylamine (3.0 eq) and 1.038 L ethyl trifluoroacetate 13a(1.3 eq). After overnight stirring, the organic mixture was distilled todryness. The resulting oil was dissolved in 15 L dichloromethane (5 mL/g11) and washed twice with 15 L 1M KH₂PO₄ and twice with 15 L de-ionizedwater. This solution was run through a 3.0 kg silica plug (1:1 silica:11), and washed with 9 L dichloromethane (3 ml/g 11, then concentratedto give a white foam. For 14a: Yield=2.9964 kg, 105%. ¹⁹F NMR (CDCl₃)□-68.7 (s).

For the preparation of 2,2-difluoropropionyl and hexafluoroisobutyrylamides, trityl piperazine succinate 11 in dichloromethane was reactedwith an aqueous solution of potassium carbonate to remove succinic acid.The dichloromethane was evaporated and the tritylpiperazine free base 12was treated with 2 eq of the ester 13c or 13d (both obtained fromSynquest, Alachua, Fla., USA) without solvent in the presence ofdiisopropylethylamine (1.0 eq). The mixture was heated at 40° C. untilcomplete. The mixture was dissolved in dichloromethane and passedthrough a plug of silica gel, eluting with ethyl acetate and heptanemixtures to provide the pure trityl piperazine amides.

Preparation of N-trifluoroacetyl piperazine, HCl salt (15): To asolution of 1.431 kg 14 in 7.16 L dichloromethane (5 mL/g 14) was addeddropwise a solution of 3.37 L 2.0 M HCl/Et₂O (2.0 eq). The reactionmixture was stirred for 1 hr, and the product was collected byfiltration. The filter cake was washed with 2.0 L dichloromethane. Thesolids were dried at 40° C. in a vacuum oven for 24 hr. For 15a:Yield=724.2 g, 98.3%. ¹⁹F NMR (CDCl₃) □ −68.2 (s); melting point=140° C.Recrystallization of a small sample from ethanol raised the meltingpoint to 154-156° C.

Preparation of Activating Agent (6): To a cooled suspension of 15 (1.0eq) in Toluene (10 mL/g 15) was added diisopropylethylamine (4.0 eq).The mixture was stirred in an ice bath for 1 hr and the salts wereremoved by filtration. The filter cake was washed twice with toluene(1.5 mL/g). The toluene solution of 15 free base (13 mL/g) was addedslowly to a ice cooled solution of POCl₃ (1.2 eq) in toluene. Thereaction mixture was stirred in an ice bath for 1 hr, then washed twicewith 1 M KH₂PO₄ (13 mL/g) and once with and de-ionized water (13 mL/g).This solution was dried over Na₂SO₄ and distilled to dryness. Theresulting amorphous solid was dissolved in dichloromethane (2 mL/g 15)and again distilled to dryness. For a 200 g batch of 15a the yield wasof 6a was 226.9 g, 75%. ¹⁹F NMR (CDCl₃) is □ −68.85 (s); ³¹P NMR (CDCl₃)□ 15.4 (s).

Preparation of Activated Subunits (7) (See FIG. 2C): To a cooledsolution/slurry of morpholino subunit 1a-e (1.0 eq) in dichloromethane(5 mL/g subunit) were added successively 2,6-lutidine (1.6 eq),N-methylimidazole (0.3 eq), and 6a-d (1.6 eq) in dichloromethane (2 ml/g6). The solution was allowed to warm to room temperature. After 3 hr,the solution was washed with 1M citric acid (pH 3). The organic layerwas dried over Na₂SO₄, the solvents removed by distillation and toluene(5 mL/g) added. The product was precipitated by dropwise addition of thesolution into heptanes (20 ml/g subunit) then collected by filtration.The crude product was purified by silica gel chromatography (gradient ofethyl acetate/heptane). The solvents were concentrated and replaced withtoluene or ethyl benzene (5 ml/g subunit). The amorphous product wasprecipitated into heptane (20 ml/g subunit) then collected byfiltration. Yield=50-70%. ¹⁹F NMR CDCl₃ shows one or two peaks withchemical shifts at about □-68.8; ³¹P NMR (CDCl₃) typically shows twosinglet peaks with chemical shifts at 13.0 to 13.4.

Example 4 Morpholino Subunits with Pro-Cationic Linkages Type (B2) (SeeFIG. 2C)

Primary Amine Containing Side Chain:

Hexamethylenediamine (100 g, 1 eq) was dissolved in methanol (1 L) andtreated dropwise with a solution of ethyl trifluoroacetate (103 mL, 1eq) in 150 mL methanol. Very slight warming of the solution occurs. Thereaction was stirred for 30 min at room temperature after addition. TLCusing chloroform/methanol/corm ammonia (8:3:1) shows the presence ofamine. The solvents were removed by rotary evaporation, and the residuedissolved in toluene/ethyl acetate (1:3, 1 L) then washed four timeswith 10% saturated aqueous sodium chloride solution to effect completeremoval of excess diamine. Evaporation yields 117 g crude 30 amine whichwas used in the activation reaction as for the piperazine example above.Crude 8a was reacted with 1a using the conditions above to give 9a. Thecorresponding reaction with the other subunits produces 9b-e. Thealternate amide protected amines were prepared and used in the samemanner as previous examples, with amides from esters 13c,d formed byreaction of the amine with neat ester.

Secondary Amine Containing Side Chain:

N,N′-Dimethylethylenediamine (36.3 mL, 3 eq.) was mixed with tritylchloride (31.8 g, 1 eq.) in dichloromethane (300 mL). After 30 min thesolvent was removed by evaporation and 300 mL toluene was added. Thesolution was washed three times with 300 mL water and finally with anequal volume of saturated aqueous sodium chloride. The foam formed onevaporation was used without purification.

The foam was dissolved in 400 mL methanol and 100 mL dichloromethane.Ethyl trifluoroacetate (17.5 mL) was added. After 30 min, the mixturewas evaporated to dryness, 300 mL dichloromethane added, and thesolution washed three times with an equal volume of water, and then oncewith saturated aqueous sodium chloride. After drying over sodiumsulfate, the organic layer was evaporated to dryness. The product waspurified by silica chromatography using 10% ethyl acetate heptanecontaining 1% lutidine to afford 24.8 g pure trityl amide.

The trityl amide was dissolved in dichloromethane (180 mL) and treateddropwise with 2 M HCl in ether (85 mL) and stirred at room temperaturefor 3 hr. The precipitated solid was filtered and dried overnight underhigh vacuum. The recovered product (10.06 g) was suspended in 100 mLdichloromethane and treated with diisopropylethylamine (25.0 mL) atwhich time a solution formed. This mixture was added to phosphorusoxychloride (4.6 mL) in toluene (100 mL) with stirring at 0° C. in anice bath. The reaction was continued 12 hr at room temperature. At thattime, the reaction was washed twice with 1 M KH₂PO₄ (100 mL), and driedover sodium sulfate. After filtration and evaporation a brown solid wasobtained that was used directly.

The brown solid was dissolved in 20 mL dichloromethane and added to asolution of Id (13.6 g) in dichloromethane (40 mL) containing2,6-lutidine (5.24 mL) and N-methylimidazole (0.672 mL). After four hrat room temperature, the reaction was washed twice with 1 M citric acidbuffer at pH=3. The solution was evaporated to dryness and the productpurified by chromatography on silica using an ethyl acetate/heptanegradient. Similar reactions afford the corresponding protectedN-methyl-N-methylaminoethyl substituted activated subunits.

Example 5 Morpholino Subunits with Pro-Cationic Linkages Type (B3) (SeeFIG. 2E)

Oxidation of 1: All glassware was oven dried overnight and cooled undervacuum or with a stream of N₂. All solutions were prepared andtransferred under N₂. The starting alcohol (1) was dried under vacuum at50° C. for 24 hr prior to use.

A solution of 1 (1 eq; 25 mmol) in DMSO/dichloromethane (1:2DMSO/dichloromethane (v:v); 5 mL/g 1) was added dropwise over 15 min tothe Swern reagent (prepared by adding DMSO (2.2 eq) to a solution ofoxalyl chloride (1.1 eq) in dichloromethane (21 mL/g) at −60° C. andstirring for 10 min). After stirring at −60° C. for 25 min,triethylamine (5 eq) was added over 10 min during which time a whiteprecipitate formed. Additional dichloromethane (5 mL/g 1) was added andthe reaction mixture stirred in a water bath for 25 min.

The reaction mixture was diluted with isopropanol/dichloromethane (1:15isopropanol/dichloromethane (v:v); 15 mL/g 1) and washed twice with 1:1(v:v) water/brine (20 ml/g 1). The solution was dried over Na₂SO₄ andconcentrated to give the aldehydes 16a-e as pale yellow foams which wereused without further purification. Yield=>100%.

Reductive Amination of 16:

A solution of methylamine acetate (10 eq; 1.16 M solution in methanol)was added to a solution of 16a-e (1 eq; 25 mmol) in methanol (8 mL/mmol16). After adjustment to pH 8 with glacial acetic acid, the reactionmixture was stirred at room temperature for 1 hr and BH₃ pyridine (2 eq)was added. After stirring for a further 1 hr, the reaction mixture wasconcentrated to a viscous oil. To the crude product dissolved indichloromethane (10 mL/mmol 16) was added 9-fluorenylmethylchloroformate (FMOC chloride) (1.5 eq) followed by diisopropylethylamine(2.5 eq) and the solution stirred at room temperature for 30 min. Thereaction mixture was diluted with dichloromethane (8 mL/mmol) and washedtwice with 1:1 (v:v) water/brine (20 ml/mmol 16). The solution was driedover Na₂SO₄, the solvent removed and the crude product was purified bysilica gel chromatography (gradient of acetone/chloroform). Yield=40-60%of 18a-e.

A solution of 18 (1 eq; 2.8 mmol) in 1%1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)/N,N-dimethylformamide (5 mL/g18) was stirred at room temperature for 30 min. The reaction mixture wasdiluted with chloroform (15 mL/g 18) and washed with 1:1 (v:v)water/brine (10 mL/g 18). The aqueous phase was re-extracted once withchloroform (10 mL/g 18), the combined organic solutions dried overNa₂SO₄ and the solvent removed. The residue was dissolved inN,N-dimethylformamide (15 mL/g 18), washed six times with hexanes (15mL/g 18) to remove/reduce the dibenzofulvene by-product and the solventremoved to give the product 17a-e as an off-white solid. Yield=85%

Freshly distilled ethyl phosphorodichloridate (3 eq) was added dropwiseover 3 min to a solution of 17 (1 eq; 2.3 mmol) anddiisopropylethylamine (3 eq) in dry dichloromethane (40 mL/g 17) underN₂ at 0° C. The cooling bath was removed and the reaction mixture wasstirred at room temperature until complete by TLC (30 min). Afterremoval of the solvent, the crude product was purified directly bysilica gel chromatography (ethyl acetate/hexane gradient). Yield=50-60%.This subunit is useful for introduction of uncharged linkages of type(a). This general method was applied below for the introduction ofcharges linkages of type (b3).

Reductive amination with a long chain ethereal diamine with the Tsubunit: 4-Methoxytriphenylmethyl chloride (15.4 g, 50 mmol) wasdissolved in toluene and added dropwise to a stirred solution of4,7,10-trioxa-1,13-tridecaneamine (150 mmol) in dichloromethanecontaining 50 mmol triethylamine. Reaction completion was convenientlyfollowed by TLC eluting with ethanol/conc ammonia (4:1, v/v) andvisualizing with ninhydrin or UV. When the reaction was done, thesolution was washed with water to remove the free amine. The solvent wasremoved by evaporation and the crude product used without purificationin the next step. Ethyl trifluoroacetate (1.5 eq) was added to asolution of methoxytritylated amine (1 eq; 25 mmol) and triethylamine(1.5 eq) in dichloromethane (12 mL/g amine) at 0° C., the cooling bathwas removed and the reaction mixture stirred at room temperature for 4hr. On reaction completion (TLC), the reaction mixture was diluted withdichloromethane (12 ml/g amine) washed twice with 1:1 (v:v) water/brine(20 ml/g 1), dried over Na₂SO₄ and concentrated to a viscous pale yellowoil. The crude methoxytritylated amide product was purified by silicagel chromatography (gradient of ethyl acetate/chloroform). Yield=60-70%.

p-Toluenesulfonic acid (1.5 eq) was added to solution ofmethoxytritylated amide (3 eq; 7.5 mmol) inmethanol/trifluoroethanol/dichloromethane (1:10:89 (v:v:v); 5 ml/g 2)and the yellow-orange solution stirred at room temperature for 30 minwhen reaction was complete (TLC). The reaction mixture was neutralizedto pH 7 with triethylamine and evaporated. The crude product wasredissolved in methanol (5 mL/g methoxytritylated amide), the pHadjusted to pH 7 if necessary and re-evaporated. The methanol additionand evaporation was repeated once more and the crude amine used withoutfurther purification.

The aldehyde 16d (1 eq; 2.5 mmol) was added to a solution of the crudeamine in methanol (5 mL/g crude amine; 12.5 mL/g 16d) and the pHadjusted to pH 8 with acetic acid. The reaction mixture was stirred atroom temperature for 1 hr and borane-pyridine (2 eq) added. The pH wasadjusted, if necessary, to maintain the starting pH and the reactionmixture stirred for 1 hr or until complete by TLC. The reaction mixturewas evaporated, the residue dissolved in dichloromethane (12.5 mL/g 16d)and 9-fluorenylmethyl chloroformate (2 eq) and diisopropylethylamine (3eq) added and the reaction mixture stirred at room temperature for 45min. The reaction mixture was partitioned between dichloromethane and1:1 (v:v) water/brine (12.5 mL/g 16d of each) and the aqueous phasere-extracted with dichloromethane (12.5 mL/g 16d). The combined organicswere washed with saturated aqueous sodium chloride (25 mL/g 16d), driedover Na₂SO₄ and the solvent removed. The crude product was purified bysilica gel chromatography (gradient of methanol/chloroform) to giveproduct 21d as a white foam. Yield=40%.

Deprotection:

A solution of crude 21d (1 eq; 0.9 mmol) in 20%triethylamine/N,N-dimethylformamide (15 mL/mmol 21d) was heated at 50°C. for 30 min when no 21d remained by TLC. The cooled reaction mixturewas extracted four times with hexanes (30 mL/mmol 21d) to remove thedibenzofulvene by-product and the solvent removed. The residue wasdissolved in isopropanol (15 mL/mmol 21d), evaporated to a foam thendissolved in a minimum volume of dichloromethane and precipitated fromhexanes (150 mL/g 21d) to give the product 20d as an off white solid.Yield=90%.

Activation:

A solution of 20d (1 eq; 0.78 mmol) and diisopropylethylamine (3 eq) wasprepared in dry dichloromethane (20 mL/g 20d) under nitrogen and addeddropwise over ˜3 min to a solution of ethyl phosphorodichloridate (3 eq)in dry dichloromethane (20 mL/g 20d) under N₂ at 0° C. The cooling bathwas removed and the reaction mixture was stirred at room temperatureuntil complete by TLC (20 min). The solution was concentrated toapproximately ½ volume and purified directly by silica gelchromatography (acetone/chloroform gradient). Yield=40-50% of 22d.

Reductive amination with a long chain ethereal diamine with the CSubunit:

Subunit 1b was oxidized by an alternative method. The subunit (5 g, 1eq) was added to a solution formed by adding pyridine (9.15 eq) thentrifluoroacetic acid (4.58 eq). The solution placed in room temperaturewater bath and stirred. When the solution was clear,diisopropylcarbodiimide (7.23 eq) was added slowly. After two hr, thesolution was added to 800 mL of saturated aqueous sodium chloridesolution. After stirring for 20 min, the mixture was filtered. Theproduct was dissolved in acetone and precipitated into de-ionized water.The filtered product was dried under vacuum. The yield was 70-80%. Theoxidized subunit may be used as is, but may be purified bychromatography on silica using ethyl acetate/dichloromethane mixtures.

The diamine 4,7,10-trioxa-1,13-tridecaneamine (33 g, 1 eq) was dissolvedin 150 mL diethyl ether, cooled to 0° C., and the solution treatedslowly with a solution of ethyl trifluoroacetate (32 g, 1.5 eq) in 50 mLether. TLC indicates reaction completion with only traces of diamineremaining.

A portion of this solution (45 mL, 3 eq amine relative to aldehyde) wasadded to a stirred solution of 3.42 g 16b in 20 mL methanol. After fivemin was added p-nitrophenol (2.52 g), and after 20 min was added sodiumcyanoborohydride (3.2 g, 8 eq). After 160 min at room temperatureadditional sodium cyanoborohydride (1.2 g) and nitrophenol (0.8 g) wereadded. The solution was poured into 800 mL of room temperature water,giving a suspension of solids and viscous oil. The water was removed bydecantation and the product dried in vacuo. The entire product wasdissolved in ethyl acetate and applied to 250 mL silica gel packed inthe same solvent. The column was washed with 2% triethylamine/ethylacetate and the product eluted with a 1% solution of triethylamine in6:1 to 10:1 ethyl acetate:ethanol mixture. The fractions containingproduct were evaporated dried in vacuo to yield 1.95 g, 37% of the amine20b.

The activated 5′-amino C subunit with the long chain ethereal side chainamine was prepared as described above for the T compound.

Example 6 Morpholino Subunits with Type (B3) Pro-Cationic Linkages byAlkylation (See FIG. 2F)

Hexamethylenediamine (100 g) was dissolved in methanol (1 L) and treateddropwise with a solution of ethyl trifluoroacetate (103 mL) in 150 mLmethanol. Very slight warming of the solution occurs. The reaction wasstirred for 30 min at room temperature after addition. TLC usingchloroform/methanol/conc ammonia (8:3:1) shows the presence of amine.The solvents were removed by rotary evaporation, the residue dissolvedin toluene/ethyl acetate (1:3, 1 L) then washed four times with 10%saturated aqueous sodium chloride solution to effect complete removal ofexcess diamine. Evaporation yields 117 g crude amine which was useddirectly with the tosylated subunit formed below.

Subunit 1b (20 g) dissolved in dichloromethane (200 mL) was treated withN-methylimidazole (11 mL) and the mixture cooled in an ice bath.p-Toluenesulfonyl chloride (8 g) was added in one step, the solutionstirred for 10 min, and the flask placed at 4° C. for 16 hr. TLC (2%methanol in dichloromethane) indicates reaction completion. The reactionwas worked up by adding 300 mL dichloromethane and washing with threetimes 300 mL of 10% saturated aqueous sodium chloride, and evaporatingto yield 23b as a foam.

The tosylate 23b (17 g) and the monoprotected amine (46.5 g, containingsome bis acylated diamine) were mixed in acetonitrile (200 mL) alongwith triethylamine (15 mL). Following 16 hr at 45° C., the mixture wasevaporated and the residue resuspended in N,N-dimethylformamide (200mL). The mixture became homogenous at 45° C. The solution was heated for5 days, at which time it was cooled to ambient temperature, and mixedwith 1 L of 10% saturated aqueous sodium chloride and 800 mL ethylacetate. The organic layer was washed with 1 L 20% saturated aqueoussodium chloride, stirred with sodium sulfate, filtered and evaporated to48 g of alkylation product, which contains a mixture of benzoylated anddebenzoylated heterocyclic base.

A portion of the crude product above (9 g) was suspended indichloromethane, cooled to 0° C., and treated with a solution ofN-(9-fluorenylmethoxycarbonyloxy)succinimide (FMOC-OSu) (ChemicalAbstracts number 82911-69-1) in 40 mL dichloromethane. The reaction wascomplete after 20 minute. To the solution was added 3.3 mLN-methylimidazole then 1.9 mL of benzoyl chloride to re-protectdebenzoylated species. After 10 more min at 0° C., the reaction wasallowed to warm to room temperature. The reaction was diluted with 150mL dichloromethane, washed with 250 mL pH=7 phosphate buffer, washedtwice with 250 mL 10% saturated aqueous sodium chloride, dried oversodium sulfate, and evaporated. The residue was loaded onto 500 mLsilica using dichloromethane (3 L), and eluted with mixtures of ethylacetate in dichloromethane (1 L each of 5%, 10%, 15%, 2 L of 20%, 2 L of40%) The last eluent provided 2.9 g of pure benzoylated FMOC protected5′-amino subunit 24b. Washing the column with 2 L of 5%methanol/dichloromethane allowed the recovery of 5.4 g of thedebenzoylated FMOC protected 5′-amino subunit.

The FMOC group was removed from the product above (7.1 g) by treatmentwith piperidine (28 mL) in DMF (140 mL). After 5 min at roomtemperature, the reaction was partitioned between dichloromethane (400mL) and water (30 mL). The organic layer was washed three times with 400mL 10% saturated aqueous sodium chloride. Evaporation provided 8.8 gcrude free amine, purified by chromatography on silica (360 mL), usingdichloromethane (1 L), 30% ethyl acetate/dichloromethane (2 L), and 5%methanol/dichloromethane (3 L) to provide 2.5 g of amine product.

One gram of this amine was dissolved in dichloromethane (10 mL) at 0° C.and treated successively with N-ethylmorpholine (500 mL), then ethylphosphorodichloridate (230 mL). Triethylamine (227 mL) was added and themixture became homogeneous. The reaction was complete after 4 hr. Afterthe usual aqueous workup the product was purified by silicachromatography (60 mL) using 10-50% ethyl acetate/heptane mixtures togive 800 mg of the activated subunit 25b.

The same process was used to make the activated T subunit with protectedhexamethylene diamine side chain at the 5′-position. Subunit Id (50 g)was reacted with p-toluenesulfonyl chloride (23.7 g) in dichloromethane(500 mL) and N-methylimidazole (16.5 mL). After one hr at 0° C. and 4 hrat room temperature, the reaction was diluted with 400 mLdichloromethane and washed with three times with 1 L of 10% saturatedaqueous sodium chloride. After drying over sodium sulfate andevaporation the residue weighed 57 g. The residue (15 g) and 40 g of thecrude mono(trifluoroacetylated)hexamethylene diamine were reacted atreflux overnight in 100 mL acetonitrile. The residue after evaporationwas dissolved in 2% methanol/dichloromethane and applied to silica. Thecolumn was eluted with dichloromethane, 50% ethylacetate/dichloromethane, ethyl acetate, 80% ethylacetate/dichloromethane, and finally 5% methanol/dichloromethane toelute the product in >98% purity. One gram of this product was activatedand purified as above to yield 300 mg (25%) of the activated subunit25d.

In a similar fashion, 1a-e were reacted with3,3′-diamino-N-methyldipropylamine, 26 which affords a side chain withtwo cationic sites as in 27a-e.

Example 7 Morpholino Subunits with Pro-Cationic Sulfamide Linkages (SeeFIG. 2G)

The 5′-methylamino subunit 17a-e (1 eq) in dimethylformamide (10 mL/g)was treated with sulfur trioxide/pyridine (4 eq), pyridine (8 eq)followed by triethylamine (6 eq). After 16 hr, the reaction was added toexcess saturated aqueous sodium chloride and the dried precipitatechromatographed on silica using 5% methanol/chloroform and containing 2%triethylamine. The triethylammonium salt of the sulfamic acid 28a-e soisolated was dissolved in dichloromethane (20 mL/g). Pyridine (3.2 eq)was added and the mixture cooled under nitrogen in a dry-ice acetonebatch. The solution was treated dropwise with 1.1 eq phosgene in toluenesolution. After 25 min, the solution was allowed to warm to roomtemperature over 20 min. The solution was rotary evaporated to an oilthat was dissolved in chloroform and directly chromatographed on silicausing 40% ethyl acetate and hexane. The product 29a-e obtained in 50%yield, was used for the introduction of sulfamide linkages of type (a).The 5′-amino subunit from hexamethylene diamine (24a-e) was deprotected,sulfated and activated in a similar fashion to provide 30a-e.

Example 8 Preparation of Disulfide Anchor (See FIG. 2H)

Preparation of symmetrical disulfide 32:1,1′-Carbonyldiimidazole (CDI)(12.402 g; 2.2 eq.) was suspended in dichloromethane (5.25 mL/g) andcooled on an ice bath. Hydroxyethyl disulfide 31 (5.36 g; 1 eq.) wasdissolved in dichloromethane (10 mL/g) and tetrahydrofuran (1 mL/g). Thediol solution was added to the CDI slowly such that the temperature ofthe mixture stayed below 4° C. for the duration of the reaction. Uponreaction completion (once addition was complete), de-ionized water (93.8μL, 0.15 eq.) was added to quench the reaction. Independently, 11 (32.59g; 2.1 eq.) was dissolved in toluene (8 mL/g 11), dichloromethane (2mL/g 11), and methanol (2 mL/g 11). K₂CO₃ (22.09 g; 4.6 eq.) wasdissolved in de-ionized water (10 mL/g). The K₂CO₃ solution added to thesolution of 11; the mixture was stirred and then separated into twolayers. The cloudy organic layer was distilled to remove 90 grams; theresulting water droplets were separated and acetone (8 mL/g 11) wasadded to the organic layer. The solution of CDI activated disulfide diolwas added to the solution of free base 12 and concentrated to 225 mL.Acetone (10 mL/g 11) was added and the mixture was concentrated to 225mL. The mixture was heated to reflux and solid began crystallizing outof solution. Upon completion, the reaction mixture was cooled and thesolid (32) was isolated by filtration. Yield: 27.92 g; 93.1% (based onweight-based assay).

Preparation of disulfide alcohol 33: 32 (36.00 g; 32.1 mmol; 1 eq.) wassuspended in acetone (2.8 mL/g 32). Hydroxyethyl disulfide (78.51 mL; 20eq.) was added followed by acetone (1.7 mL/g 32). 5% NaOH/methanol (2.85mL; 0.1 eq.) was added; the pH of the mixture was 10 by pH paper.Triphenylphosphine (8.42 g; 1 eq.) was added followed by acetone (1.1mL/g 32). All solids went into solution and then product began tocrystallize out. After sixteen hr, the reaction mixture was neutralizedwith acetic acid (2.4 g; 0.2 eq.). The crude product was isolated byfiltration. The crude solid 33 was subjected to two refluxing acetonereslurries (5 mL/g).

After filtration the crude product was suspended in dichloromethane(7.25 mL/g 32). The mixture was heated until a clear solution formed(35° C.). The solution was extracted five times with an equal volume ofde-ionized water and the final organic layer was concentrated to 155 mL.Dichloromethane was added (4.3 mL/g 32), and the solution was againconcentrated to 155 mL. CDI (9.17 g; 1.1 eq.) was added and the mixturewas stirred at room temperature. Upon reaction completion (˜20 min) thereaction mixture was washed twice with an equal volume of de-ionizedwater, then ethylbenzene (2.1 mL/g 32) was added. The solution wasconcentrated to 65.2 g, reducing the dichloromethane in the solution to0.17%, and stirred on an ice bath to crystallize the product. Theproduct 34 was isolated by filtration. Yield: 44%.

Example 9 Triethylene Glycol Tail (See FIG. 2I)

Preparation of trityl piperazine phenyl carbamate 35: To a cooledsuspension of compound II in dichloromethane (6 mL/g 11) was added asolution of potassium carbonate (3.2 eq) in water (4 mL/g potassiumcarbonate). To this two-phase mixture was slowly added a solution ofphenyl chloroformate (1.03 eq) in dichloromethane (2 g/g phenylchloroformate). The reaction mixture was warmed to 20° C. Upon reactioncompletion (1-2 hr), the layers were separated. The organic layer waswashed with water, and dried over anhydrous potassium carbonate. Theproduct 35 was isolated by crystallization from acetonitrile. Yield=80%

Preparation of carbamate alcohol 36: Sodium hydride (1.2 eq) wassuspended in 1-methyl-2-pyrrolidinone (32 mL/g sodium hydride). To thissuspension were added triethylene glycol (10.0 eq) and compound 35 (1.0eq). The resulting slurry was heated to 95° C. Upon reaction completion(1-2 hr), the mixture was cooled to 20° C. To this mixture was added 30%dichloromethane/methyl tert-butyl ether (v:v) and water. Theproduct-containing organic layer was washed successively with aqueousNaOH, aqueous succinic acid, and saturated aqueous sodium chloride. Theproduct 36 was isolated by crystallization from dichloromethane/methyltert-butyl ether/heptane. Yield=90%.

Preparation of Tail acid 37: To a solution of compound 36 intetrahydrofuran (7 mL/g 36) was added succinic anhydride (2.0 eq) andDMAP (0.5 eq). The mixture was heated to 50° C. Upon reaction completion(5 hr), the mixture was cooled to 20° C. and adjusted to pH 8.5 withaqueous NaHCO₃. Methyl tert-butyl ether was added, and the product wasextracted into the aqueous layer. Dichloromethane was added, and themixture was adjusted to pH 3 with aqueous citric acid. Theproduct-containing organic layer was washed with a mixture of pH=3citrate buffer and saturated aqueous sodium chloride. Thisdichloromethane solution of 37 was used without isolation in thepreparation of compound 38.

Preparation of 38: To the solution of compound 37 was addedN-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB) (1.02 eq),4-dimethylaminopyridine (DMAP) (0.34 eq), and then1-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (1.1eq). The mixture was heated to 55° C. Upon reaction completion (4-5 hr),the mixture was cooled to 20° C. and washed successively with 1:1 0.2 Mcitric acid/brine and brine. The dichloromethane solution underwentsolvent exchange to acetone and then to N,N-dimethylformamide, and theproduct was isolated by precipitation from acetone/N,N-dimethylformamideinto saturated aqueous sodium chloride. The crude product was reslurriedseveral times in water to remove residual N,N-dimethylformamide andsalts. Yield=70% of 38 from compound 36. Introduction of the activated“Tail” onto the disulfide anchor-resin was performed in NMP by theprocedure used for incorporation of the subunits during solid phasesynthesis.

Example 10 Preparation of the Solid Support for Synthesis of MorpholinoOligomers (FIG. 2J) Example 10a: Preparation ofAminomethylpolystyrene-Disulfide Resin

This procedure was performed in a silanized, jacketed peptide vessel(custom made by ChemGlass, NJ, USA) with a coarse porosity (40-60 μm)glass frit, overhead stirrer, and 3-way Teflon stopcock to allow N₂ tobubble up through the frit or a vacuum extraction. Temperature controlwas achieved in the reaction vessel by a circulating water bath.

The resin treatment/wash steps in the following procedure consist of twobasic operations: resin fluidization and solvent/solution extraction.For resin fluidization, the stopcock was positioned to allow N₂ flow upthrough the frit and the specified resin treatment/wash was added to thereactor and allowed to permeate and completely wet the resin. Mixing wasthen started and the resin slurry mixed for the specified time. Forsolvent/solution extraction, mixing and N₂ flow were stopped and thevacuum pump was started and then the stopcock was positioned to allowevacuation of resin treatment/wash to waste. All resin treatment/washvolumes were 15 mL/g of resin unless noted otherwise.

To aminomethylpolystyrene resin (100-200 mesh; ˜1.0 mmol/g N₂substitution; 75 g, 1 eq, Polymer Labs, UK, part #1464-X799) in asilanized, jacketed peptide vessel was added 1-methyl-2-pyrrolidinone(NMP; 20 ml/g resin) and the resin was allowed to swell with mixing for1-2 hr. Following evacuation of the swell solvent, the resin was washedwith dichloromethane (2×1-2 min), 5% diisopropylethylamine in 25%isopropanol/dichloromethane (2×3-4 min) and dichloromethane (2×1-2 min).After evacuation of the final wash, the resin was fluidized with asolution of disulfide anchor 34 in 1-methyl-2-pyrrolidinone (0.17 M; 15mL/g resin, ˜2.5 eq) and the resin/reagent mixture was heated at 45° C.for 60 hr. On reaction completion, heating was discontinued and theanchor solution was evacuated and the resin washed with1-methyl-2-pyrrolidinone (4×3-4 min) and dichloromethane (6×1-2 min).The resin was treated with a solution of 10% (v/v) diethyl dicarbonatein dichloromethane (16 mL/g; 2×5-6 min) and then washed withdichloromethane (6×1-2 min). The resin 39 was dried under a N₂ streamfor 1-3 hr and then under vacuum to constant weight (±2%). Yield:110-150% of the original resin weight.

Example 10b: Determination of the Loading ofAminomethylpolystyrene-Disulfide Resin

The loading of the resin (number of potentially available reactivesites) is determined by a spectrometric assay for the number oftriphenylmethyl (trityl) groups per gram of resin.

A known weight of dried resin (25±3 mg) is transferred to a silanized 25ml volumetric flask and ˜5 mL of 2% (v/v) trifluoroacetic acid indichloromethane is added. The contents are mixed by gentle swirling andthen allowed to stand for 30 min. The volume is brought up to 25 mL withadditional 2% (v/v) trifluoroacetic acid in dichloromethane and thecontents thoroughly mixed. Using a positive displacement pipette, analiquot of the trityl-containing solution (500 μL) is transferred to a10 mL volumetric flask and the volume brought up to 10 mL withmethanesulfonic acid.

The trityl cation content in the final solution is measured by UVabsorbance at 431.7 nm and the resin loading calculated in trityl groupsper gram resin (μmol/g) using the appropriate volumes, dilutions,extinction coefficient (E: 41 μmol⁻¹ cm⁻¹) and resin weight. The assayis performed in triplicate and an average loading calculated.

The resin loading procedure in this example will provide resin with aloading of approximately 500 μmol/g. A loading of 300-400 in μmol/g wasobtained if the disulfide anchor incorporation step is performed for 24hr at room temperature.

Example 10c: Tail Loading

Using the same setup and volumes as for the preparation ofaminomethylpolystyrene-disulfide resin, the Tail can be introduced intothe molecule. For the coupling step, a solution of 38 (0.2 M) in NMPcontaining 4-ethylmorpholine (NEM, 0.4 M) was used instead of thedisulfide anchor solution. After 2 hr at 45° C., the resin 39 was washedtwice with 5% diisopropylethylamine in 25% isopropanol/dichloromethaneand once with DCM. To the resin was added a solution of benzoicanhydride (0.4 M) and NEM (0.4 M). After 25 min, the reactor jacket wascooled to room temperature, and the resin washed twice with 5%diisopropylethylamine in 25% isopropanol/dichloromethane and eight timeswith DCM. The resin 40 was filtered and dried under high vacuum. Theloading for resin 40 is defined to be the loading of the originalaminomethylpolystyrene-disulfide resin 39 used in the Tail loading.

Example 11 Preparation of Morpholino Oligomers on an AutomatedSynthesizer Example 11a: Solid Phase Synthesis

Morpholino Oligomers were prepared on a Gilson AMS-422 Automated PeptideSynthesizer in 2 mL Gilson polypropylene reaction columns (Part#3980270). An aluminum block with channels for water flow was placedaround the columns as they sat on the synthesizer. The AMS-422 willalternatively add reagent/wash solutions, hold for a specified time, andevacuate the columns using vacuum.

For oligomers in the range up to about 25 subunits in length,aminomethylpolystyrene-disulfide resin with loading near 500 μmol/g ofresin is preferred. For larger oligomers, aminomethylpolystyrene-disulfide resin with loading of 300-400 μmol/g of resin is preferred. If amolecule with 5′-Tail is desired, resin that has been loaded with Tailis chosen with the same loading guidelines.

The following reagent solutions were prepared:

Detritylation Solution: 10% Cyanoacetic Acid (w/v) in 4:1dichloromethane/acetonitrile; Neutralization Solution: 5%Diisopropylethylamine in 3:1 dichloromethane/isopropanol; CouplingSolution: 0.18 M (or 0.24 M for oligomers having grown longer than 20subunits) activated Morpholino Subunit of the desired base and linkagetype and 0.4 M N-ethylmorpholine, in 1,3-dimethylimidazolidinone.Dichloromethane (DCM) was used as a transitional wash separating thedifferent reagent solution washes.

On the synthesizer, with the block set to 42° C., to each columncontaining 30 mg of aminomethylpolystyrene-disulfide resin (or Tailresin) was added 2 mL of 1-methyl-2-pyrrolidinone and allowed to sit atroom temperature for 30 min. After washing with 2 times 2 mL ofdichloromethane, the following synthesis cycle was employed:

Step Volume Delivery Hold time Detritylation 1.5 mL Manifold 15 secondsDetritylation 1.5 mL Manifold 15 seconds Detritylation 1.5 mL Manifold15 seconds Detritylation 1.5 mL Manifold 15 seconds Detritylation 1.5 mLManifold 15 seconds Detritylation 1.5 mL Manifold 15 secondsDetritylation 1.5 mL Manifold 15 seconds DCM 1.5 mL Manifold 30 secondsNeutralization 1.5 mL Manifold 30 seconds Neutralization 1.5 mL Manifold30 seconds Neutralization 1.5 mL Manifold 30 seconds Neutralization 1.5mL Manifold 30 seconds Neutralization 1.5 mL Manifold 30 secondsNeutralization 1.5 mL Manifold 30 seconds DCM 1.5 mL Manifold 30 secondsCoupling 350 uL-500 uL Syringe 40 minutes DCM 1.5 mL Manifold 30 secondsNeutralization 1.5 mL Manifold 30 seconds Neutralization 1.5 mL Manifold30 seconds DCM 1.5 mL Manifold 30 seconds DCM 1.5 mL Manifold 30 secondsDCM 1.5 mL Manifold 30 seconds

The sequences of the individual oligomers were programmed into thesynthesizer so that each column receives the proper coupling solution(A,C,G,T,I) in the proper sequence. When the oligomer in a column hadcompleted incorporation of its final subunit, the column was removedfrom the block and a final cycle performed manually with a couplingsolution comprised of 4-methoxytriphenylmethyl chloride (0.32 M in DMI)containing 0.89 M 4-ethylmorpholine.

Example 11b: Cleavage from the Resin and Removal of Bases and BackboneProtecting Groups

After methoxytritylation, the resin was washed 8 times with 2 mL1-methyl-2-pyrrolidinone. One mL of a cleavage solution consisting of0.1 M 1,4-dithiothreitol (DTT) and 0.73 M triethylamine in1-methyl-2-pyrrolidinone was added, the column capped, and allowed tosit at room temperature for 30 min. After that time, the solution wasdrained into a 12 mL Wheaton vial. The greatly shrunken resin was washedtwice with 300 μL of cleavage solution. To the solution was added 4.0 mLconc aqueous ammonia (stored at −20° C.), the vial capped tightly (withTeflon lined screw cap), and the mixture swirled to mix the solution.The vial was placed in a 45° C. oven for 16-24 hr to effect cleavage ofbase and backbone protecting groups.

Example 11c: Initial Oligomer Isolation

The vialed ammonolysis solution was removed from the oven and allowed tocool to room temperature. The solution was diluted with 20 mL of 0.28%aqueous ammonia and passed through a 2.5×10 cm column containingMacroprep HQ resin (BioRad). A salt gradient (A: 0.28% ammonia with B: 1M sodium chloride in 0.28% ammonia; 0-100% B in 60 min) was used toelute the methoxytrityl containing peak. The combined fractions werepooled and further processed depending on the desired product.

Example 11d: Demethoxytritylation of Morpholino Oligomers: MethoxytritylOff Oligomers

The pooled fractions from the Macroprep purification were treated with 1M H₃PO₄ to lower the pH to 2.5. After initial mixing, the samples sat atroom temperature for 4 min, at which time they are neutralized to pH10-11 with 2.8% ammonia/water. The products were purified by solid phaseextraction (SPE).

Amberchrome CG-300M (Rohm and Haas; Philadelphia, Pa.) (3 mL) is packedinto 20 mL fritted columns (BioRad Econo-Pac Chromatography Columns(732-1011)) and the resin rinsed with 3 mL of the following: 0.28%NH₄OH/80% acetonitrile; 0.5M NaOH/20% ethanol; water; 50 mM H₃PO₄/80%acetonitrile; water; 0.5 NaOH/20% ethanol; water; 0.28% NH₄OH.

The solution from the demethoxytritylation was loaded onto the columnand the resin rinsed three times with 3-6 mL 0.28% aqueous ammonia. AWheaton vial (12 mL) was placed under the column and the product elutedby two washes with 2 mL of 45% acetonitrile in 0.28% aqueous ammonia.The solutions were frozen in dry ice and the vials placed in a freezedryer to produce a fluffy white powder. The samples were dissolved inwater, filtered through a 0.22 micron filter (Pall Life Sciences,Acrodisc 25 mm syringe filter, with a 0.2 micron HT Tuffryn membrane)using a syringe and the Optical Density (OD) was measured on a UVspectrophotometer to determine the OD units of oligomer present, as wellas dispense sample for analysis. The solutions were then placed back inWheaton vials for lyophilization.

Example 11e: Analysis of Morpholino Oligomers

MALDI-TOF mass spectrometry was used to determine the composition offractions in purifications as well as provide evidence for identity(molecular weight) of the oligomers. Samples were run following dilutionwith solution of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid),3,4,5-trihydroxyacetophenone (THAP) or alpha-cyano-4-hydroxycinnamicacid (HCCA) as matrices.

Cation exchange (SCX) HPLC was performed using a Dionex ProPac SCX-10,4×250 mm column (Dionex Corporation; Sunnyvale, Calif.) using 25 mM pH=5sodium acetate 25% acetonitrile (Buffer A) and 25 mM pH=5 sodium acetate25% acetonitrile 1.5 M potassium chloride (buffer B) (Gradient 10-100% Bin 15 min) or 25 mM KH₂PO₄ 25% acetonitrile at pH=3.5 (buffer A) and 25mM KH₂PO₄ 25% acetonitrile at pH=3.5 with 1.5 M potassium chloride(buffer B) (Gradient 0-35% B in 15 min). The former system was used forpositively charged oligomers that do not have a peptide attached, whilethe latter was used for peptide conjugates.

Example 11f: Purification of Morpholino Oligomers by Cation ExchangeChromatography

The sample is dissolved in 20 mM sodium acetate, pH=4.5 (buffer A) andapplied to a column of Source 30 cation exchange resin (GE Healthcare)and eluted with a gradient of 0.5 M sodium chloride in 20 mM sodiumacetate and 40% acetonitrile, pH=4.5 (buffer B). The pooled fractionscontaining product are neutralized with conc aqueous ammonia and appliedto an Amberchrome SPE column. The product is eluted, frozen, andlyophilized as above.

The following oligomers exemplify this method:

5′-(EG3)-G+TGC+TCA+TGG+TGCACGG+T-3′-(H) (SEQ ID NO: 113), calculated[M+H]⁺⁼6860.9 daltons, m found [M+H]⁺⁼6861.7 daltons, useful for HCV.

5′-(EG3)-GCC+ATGGT+TTT+TTC+TC+AGG-3′-(H) (SEQ ID NO: 108), calculated[M+H]⁺⁼6825.9 daltons, found [M+H]⁺⁼6827.1 daltons, useful for Ebola.

5′-(EG3)-+TGGGT+ATG+TTGT+AGCC+AT-3′-(H) (SEQ ID NO: 109), calculated[M+H]⁺⁼7245.2 daltons, found [M+H]⁺⁼7246.8 daltons, useful for Ebola.

5′-(EG3)-CC+TGCCC+TTTGT+TCT+AGT+TG-3′-(H) (SEQ ID NO: 110), calculated[M+H]⁺⁼7092.2 daltons, found [M+H]⁺⁼7093.8 daltons, useful for Ebola.

Example 11g: 3-′-Methoxytrityl and 3′ Trityl Morpholino Oligomers

The Macroprep purified oligomers were directly applied to the solidphase extraction columns, and the 3′-methoxytritylated oligomers wereisolated and quantified in the same manner as the demethoxytritylatedspecies.

Example 12 SYNTHESIS OF N2,O6-PROTECTED MORPHOLINO G (DPG) FOR LARGESCALE OLIGOMER Synthesis (FIG. 2K)

Preparation of 41: To a cooled solution of 1c and imidazole (1.3 eq) indichloromethane (8 mL/g 1) was added a solution oftert-butyldimethylchlorosilane (1.2 eq) in dichloromethane. Afteraddition, the solution was warmed to 20° C. Upon reaction completion(1-3 hours), this solution was washed successively with 1 M citratebuffer (adjusted to pH 3 with NaOH) and water. The resulting organicsolution was distilled to azeotropically remove water and used directlyin the next step.

Preparation of 42: To a 0° C. cooled solution of 41 in dichloromethanewere added successively triethylamine (1.2 eq), 4-dimethylaminopyridine(0.1 eq), and triisopropylbenzenesulfonyl chloride (1.1 eq). Thesolution was warmed to 20° C. Upon reaction Completion (3-9 hours), thesolution was washed successively with 1 M KH₂PO₄ and water. Theresulting organic solution was distilled to azeotropically remove waterand used directly in the preparation of compound 44.

Preparation of 43: To a solution of 4-hydroxybenzaldehyde (1.0 eq) andN-methylimidazole (0.2 eq) in toluene was added a solution of KHCO₃ (2.0eq) in water. To the resulting two-phase mixture was addedtrimethylacetyl chloride (1.4 eq). Upon reaction completion (1-2 hours),methanol (1.0 eq) was added, and the mixture was stirred for 1 hour.After separation of layers, the organic layer was washed successivelywith 1 M KH₂PO₄ and water. The resulting organic solution was distilledto azeotropically remove water and diluted with THF. To this solutionwas added 5% Pd/C catalyst (0.004 eq, Johnson Matthey, West Deptford,N.J., USA), and the mixture was hydrogenated under 5-30 psi H₂. Uponreaction completion (4-8 hours), the mixture was filtered through a padof Celite and washed with pH 6.5 phosphate buffer. The product wascrystallized from toluene/heptane. Yield=80%.

Preparation of 44: To a cooled solution of 3 in dichloromethane wasadded N-methylpyrrolidine (2.0 eq). After 10 minutes, 3a (1.2 eq) wasadded, followed by DBU (1.2 eq). After reagent addition, the solutionwas warmed to 20° C. Upon reaction completion (1-9 hours), the solutionwas washed successively with 1 M KH₂PO₄ and water. The resulting organicsolution was distilled to azeotropically remove water and used directlyin the next step.

Preparation of 45: To the solution of 44 in dichloromethane was addedtriethylamine trihydrofluoride (2.0 eq). Upon reaction completion (4-20hours), the solution was washed successively with sodium bicarbonatesolution, pH 6.5 phosphate buffer, and water. The resulting solution wasdistilled to remove dichloromethane, and the product was crystallizedfrom THF/water. Yield=70% from 1c.

Preparation of 46: Compound 45 was dissolved in dichloromethane (6 mL/g45) and cooled to <5° C. To this solution were added 2,6-lutidine (1.6eq), N-methylimidazole (0.3 eq), andN,N-dimethylphosphoramidodichloridate (1.6 eq). The solution was warmedto 20° C. Upon reaction completion (6-12 hours), this mixture was washedwith a pH 3 citrate buffer. The crude product was isolated byprecipitation/reslurry. The doubly protected (DPG) product 46 waspurified by silica gel chromatography (gradient of ethylacetate/heptane) and isolated by precipitation into heptane.Yield=40-60%.

Example 13 Large Scale Synthesis of Morpholino Oligomers

The reactor design for the loading of anchor and Tail onaminomethylpolystyrene resin is used for larger scale synthesis ofMorpholino Oligomers. Resin loading guidelines are the same as for thesmaller scale synthesis.

Example 13a: Solid Phase Synthesis

Protected oligomers were prepared manually by solid phase oligomersynthesis on aminomethylpolystyrene-disulfide resin (˜500 μmol/gloading) at 10 g scale (starting resin weight). Solutions used were asfollows: detritylation solution: 2% 4-cyanopyridinium trifluoroacetate(CYTFA) (w/v) in 20% trifluoroethanol/dichloromethane with 1% ethanol;neutralization solution: 5% diisopropylethylamine in 25%isopropanol/dichloromethane; coupling solution: 0.165 M (for 46 (DPG)and 5d or other T subunits) or 0.18 M (for 5a and 5b or other NCsubunits) activated Morpholino Subunit and 0.4 M N-ethylmorpholine in1,3-dimethylimidazolidinone (DMI).

After transfer of the resin to the synthesis reactor and prior toinitiating synthesis cycles, 1-methyl-2-pyrrolidinone (NMP, 20 mL/gresin) was added and allowed to sit for 1-2 hrs. After washing 2 timeswith dichloromethane (10 mL/g resin), the following synthesis cycle wasused with addition of the appropriate coupling solution of activatedMorpholino Subunit of the desired base and desired linkage type at eachcycle to give the proper sequence.

Step Volume (mL/g of starting resin)* Time (min) DCM 10-30 1-2 DCM 10-301-2 Detritylation A 10-30 2-3 Detritylation A 10-30 2-3 Detritylation A10-30 2-3 Detritylation A 10-30 2-3 Detritylation A 10-30 2-3Neutralization A 10-30 3-4 Neutralization A 10-30 3-4 Neutralization A10-30 3-4 Neutralization A 10-30 3-4 DCM 10-30 1-2 DCM 10-30 1-2Coupling   7-12** 90 Neutralization A 10-30 1-2 Neutralization A 10-301-2 Neutralization A 10-30 1-2 Neutralization A 10-30 1-2 DCM 10-30 1-2*Wash volumes are incremented to account for resin swelling; volume is10 mL/g of actual resin volume at each cycle **Coupling volumes aresufficient to maintain good mixing and are incremented to account forresin swelling

After incorporation of the final subunit, a final cycle(methoxytritylation) was performed with 0.32 M 4-methoxytriphenylmethylchloride and 0.4 M N-ethylmorpholine in DMI. After methoxytritylation,the resin was washed 8 times with NMP and then treated with cleavagesolution consisting of 0.1 M 1,4-dithiothreitol (DTT) and 0.73 Mtriethylamine in NMP (27 mL/g starting resin) for 30 min. Aftercollection of the protected oligomer solution, the resin (significantlyreduced in volume) was washed with two additional portions of cleavagesolution (13 mL/g starting resin for 15 min each) and the washes werecombined with the bulk solution. To the protected oligomer solution inan appropriately sized pressure bottle with Teflon plug (Ace Glass, NJ,USA) was added concentrated aqueous ammonia (106 mL/g starting resin,previously cooled to −20° C.), the bottle sealed, and the contents mixedby swirling. The bottle was placed in a 45° C. oven for 16-20 hr toremove base and backbone protecting groups.

Following ammonolysis, the crude oligomer solution is cooled to roomtemperature and then diafiltered against 0.28% aqueous ammonia using aPLBC 3 kd Regenerated Cellulose membrane (Millipore) to remove solventsand small molecules prior to ion exchange chromatography.

Example 13b: Purification of Morpholino Oligomers by Anion ExchangeChromatography

The crude oligomer solution obtained from diafiltration is adjusted topH 11-11.5 and loaded onto a column of ToyoPearl Super-Q 650S anionexchange resin (Tosoh Bioscience. The methoxytritylated oligomer waseluted with a gradient of 5-35% B over 17 column volume (Buffer A: 10 mMsodium hydroxide; Buffer B: 1 M sodium chloride in 10 mM sodiumhydroxide) and fractions of acceptable purity (anion exchange HPLC andmass spec) pooled.

Example 13c: Demethoxytritylation of Morpholino Oligomers

To the pooled fractions from anion exchange chromatography was addedacetonitrile (10% by volume) followed by 2 M H₃PO₄ to adjust the pH to3. The solution was mixed for 45 min and then neutralized withconcentrated aqueous ammonia to pH 7. The oligomer solution wasdiafiltered against 20 mM sodium acetate using a PLBC 3 kd RegeneratedCellulose membrane (Millipore) to exchange buffers prior to cationexchange chromatography.

Example 13d: Purification of Morpholino Oligomers by Cation ExchangeChromatography

The oligomer solution was adjusted to pH 4.5 with acetic acid and loadedonto a column of Source 30S cation exchange resin (GE Healthcare). Theoligomer was eluted with a gradient of 0-35% B over 17 column volumes(Buffer A: 20 mM sodium acetate, 25% acetonitrile, pH 4.5; Buffer B: 0.5M sodium chloride, 20 mM sodium acetate, 25% acetonitrile, pH 4.5) andfractions of acceptable purity (cation exchange HPLC and mass spec)pooled.

Example 13e: Isolation of Morpholino Oligomers

The purified oligomer solution was diafiltered against 0.028% aqueousammonia using a PLBC 3 kd Regenerated Cellulose membrane (Millipore) toremove salt and generate the oligomer free base. The desalted oligomersolution was then frozen and lyophilized to give the oligomer as a whitefluffy powder (˜12% water content). By this method compounds useful inEbola treatment were prepared:

(SEQ ID NO: 108) 5′-(EG3)-GCC + ATGGT + TTT + TTC + TC + AGG-3′-(H), 8.4 g (SEQ ID NO: 110) 5′-(EG3)-CC + TGCCC + TTTGT + AGT +TG-3′-(H), 10.0 g

Identical to the compounds made by small scale.

Example 13f: Analysis of Morpholino Oligomers by Anion Exchange HPLC

Anion exchange (SAX) HPLC was performed using a Dionex DNAPac, 4×250 mmcolumn (Dionex Corporation; Sunnyvale, Calif.) using 20 mM sodiumchloride, 10 mmol sodium hydroxide (Buffer A) and 1 M sodium chloride,10 mmol sodium hydroxide (buffer B), (Gradient 10-53% B in 20 min).

Example 14 Introduction of a Guanidinium Group into a MorpholinoOligomer Example 14a: By Direct Guanylation of Amines

10 μmol of a Morpholino Oligomer, the backbone of which contained threepiperazine secondary amines, were dissolved in 0.5 M Na₂CO₃ at 75 mg/mL.700 μmol of 1-H-pyrazole-1-carboxamidine HCl were added to theMorpholino Oligomer solution and the reaction stirred at roomtemperature. After three hours had elapsed, the reaction was dilutedwith water and purified by solid phase extraction (SPE) using anAmberchrom CG300M (Rohm and Haas; Philadelphia, Pa.) column. The SPEpurification entailed loading the sample onto the column at 20 mg/mL,washing the column with 4 column volumes of 1 M NaCl and then 3 columnvolumes of water. The product was eluted by washing the column with 3column volumes of acetonitrile/water (1:1 v/v). The product, as the HClsalt, was then lyophilized.

Strong cation exchange (SCX) purification at pH=9 of morpholinooligomers containing guanidine backbone moieties may be achieved whenthe oligomer contains at least three guanidine groups on the backboneand/or termini. Prior to lyophilization, the desalted solution fromabove was purified by SCX at pH 9 to separate the guanidine-modifiedproduct from any remaining underivatized piperazine precursor. Theliquid chromatography column had dimensions of 10×62 mm (Bio-ChemValve/Omnifit, Cambridge, United Kingdom) and contained Source 15Sstrong cation exchange media (GE Healthcare Bio-Sciences Corp.,Piscataway, N.J.). The mobile phase compositions were A) 25 mM Tris HCl,25% acetonitrile (v/v); pH 9.0 and B) 1.0 M sodium chloride, 25 mM TrisHCl, 25% acetonitrile (v/v); pH 9.0. A linear velocity of 342 cm/hr wasused. After equilibration of the column with four column volumes ofmobile phase A, the oligomer sample was loaded in mobile phase A at aconcentration of approximately 5 mg/mL. The column was then washed fortwo minutes with mobile phase A, after which fraction collection wasinitiated concurrently with, a linear gradient of 0-20% mobile phase Bover twenty-two minutes. Fractions were analyzed individually byMALDI-TOF MS. Selected fractions were pooled and desalted by solid phaseextraction. The fraction pool was diluted 5-fold with water and loadedonto an Amberchrom CG300M column. The SPE desalt entailed loading thesample onto the column at 20 mg/mL and washing the column with 3 columnvolumes of water. The product was then eluted by washing the column with3 column volumes of acetonitrile/water (1:1 v/v). The product waslyophilized and analyzed by MALDI-TOF MS and SCX HPLC.

The following oligomers exemplify this method:

5′-(EG3)-CTGGG+ATG+AG+ATCC+ATC+ACT-3′-(H) (SEQ ID NO: 112) was preparedusing resin with Tail. A sample (1000 OD) was converted by the methodsabove into:

5′-(EG3)-CTGGG(Gupip)ATG(Gupip)AG(Gupip)ATCC(Gupip)ATC(Gupip)ACT-3′-(Gu)calculated [M+H]⁺⁼7421.5 daltons. The crude product (907 OD) recoveredafter SPE, found [M+H]⁺⁻7422.6 daltons, was purified on SCX ion exchangeat pH=9 to provide 378 OD product, found [M+H]⁺⁼7420.9.

Example 14b: Introduction of Guanidinium Groups by Reaction withGuanidino Amino Acid Trifluoroacetate Salts

Preparation of Guanidino Acid Trifluoroacetate Salts.

The guanidino acid was dissolved in trifluoroacetic acid at aconcentration of 330 mg/mL with gentle heating and stirring. Once fullydissolved, the solution was added dropwise to a tenfold volumetricexcess of diethyl ether to precipitate the trifluoroacetate salt of theguanidino acid. The suspension was centrifuged, the supernatantdecanted, and the solid triturated in a fresh portion of diethyl ether.The suspension was again centrifuged, the supernatant decanted, and thesolid dried under vacuum.

Conjugation of Guanidino Acids (General) to Amines of a MorpholinoOligomer.

The morpholino oligomer, containing free amino groups on the backboneand/or termini, was dissolved in DMSO at 75 mg/mL. Separately, anactivated guanidino acid solution was prepared by dissolving thetrifluoroacetate or hydrochloride salt of the guanidino acid (2equivalents with respect to Morpholino Oligomer amines) and HBTU (1.95equivalents with respect amines) in NMP at 100 mg/mL (with respect toguanidino acid). DIEA (3 equivalents with respect to guanidino acid) wasthen added to the guanidino acid solution. The activated guanidino acidsolution was mixed briefly and immediately added to the MorpholinoOligomer solution. After three hours of stirring at room temperature,the reaction was diluted 2.33-fold with cold concentrated ammoniumhydroxide. As required, TFE was added slowly with gentle heating andmixing to redissolve the precipitated solid. The reaction was thenheated at 45° C. in a sealed vessel for 18 hours, after which it wasdiluted 15-fold with water and purified by SPE using an AmberchromCG300M (Rohm and Haas; Philadelphia, Pa.) column. The SPE purificationentailed loading the sample onto the column at 20 mg/mL, washing thecolumn with 4 column volumes of 1 M NaCl and then 3 column volumes ofwater. The product was eluted by washing the column with 3 columnvolumes of acetonitrile/water (1:1 v/v). The conjugate was lyophilizedand analyzed by MALDI-TOF MS and SCX HPLC.

The following oligomers exemplify this method:

5′-(H)-C+TTCGA+TAG+TG-3′-(H) (SEQ ID NO: 66) was prepared from usingresin with no Tail. A sample (970 OD) was converted with4-guanidinobutanoic acid by the methods above into:5′-(GuBu)-C(GuBupip)TTCGA(GuBupip)TAG(GuBupip)TG-3′-(GuBu), calculated[M+H]⁺⁼4541.2 daltons. The crude product (820 OD) recovered after SPE,found [M+H]⁺⁻4541.9 daltons, was purified on SCX ion exchange at pH=9 toprovide 356 OD product, found [M+H]⁺⁼4542.1.

Conjugation of 6-Guanidinohexanoic Acid to Secondary Amines of aMorpholino Oligomer

6-Guanidinohexanoic acid was obtained from AlzChem; Trostberg, Germany.The Morpholino Oligomer (20 μmol), an 18-mer with eight secondary aminesincorporated into the backbone (160 μmol of amine groups), was dissolvedin dimethylsulfoxide (DMSO) at 75 mg/mL. Separately, an activated6-guanidinohexanoic acid solution was prepared by dissolving 320 μmol (2molar equivalents with respect to amines) of the trifluoroacetate saltof 6-Guanidinohexanoic acid and 312 μmol of2-(1-H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate(HBTU) in 920 μL of 1-methyl-2-pyrrolidinone (NMP) and adding 960 μmolof N,N-diisopropylethylamine (DIEA). Immediately after addition of DIEA,the activated guanidino-acid solution was added to the MorpholinoOligomer solution. After stirring under nitrogen at room temperature forthree hours, 4 mL of concentrated ammonium hydroxide were added to thereaction. 7 mL of 2,2,2-trifluoroethanol (TFE) were added with gentlyheating and mixing to redissolve the precipitated solid and the solutionheated at 45° C. for 18 hours. The solution was then diluted to 200 mLwith water and purified by solid phase extraction (SPE) using anAmberchrom CG300M column as detailed above. The conjugate waslyophilized and analyzed by MALDI-TOF MS and SCX HPLC.

The following oligomers exemplify this method:

5′-(H)-C+TTCGA+TAG+TG-3′-(H) (SEQ ID NO: 66) was prepared using resinwith no Tail. A sample (635 OD) was converted by the methods above into:

5′-(GuAhx)-C(GuX)TTCGA(GuX)TAG(GuX)TG-3′-(GuAhx), calculated[M+H]⁺⁼4681.4 daltons. The crude product (563 OD) recovered after SPE,found [M+H]⁺⁻4681.2 daltons, was purified on SCX ion exchange at pH=6.5to provide 427 OD product of 93.3% purity by SCX HPLC, found[M+H]⁺⁼4682.4.

5′-(EG3)-CTGGG+ATG+AG+ATCC+ATC+ACT-3′-(H) (SEQ ID NO: 111) was preparedusing resin with Tail. A sample (1500 OD) was converted by the methodsabove into:

5′-(EG3)-CTGGG(GuX)ATG(GuX)AG(GuX)ATCC(GuX)ATC(GuX)ACT-3′-(GuAhx),calculated [M+H]⁺⁼8100.5 daltons. The crude product (1486 OD) recoveredafter SPE. found [M+H]⁺⁼8100.4 daltons, was purified on SCX ion exchangeat pH=9 to provide 700 OD product, found [M+H]⁺⁼8100.6.

This method was also used to add peptide acids such as AcRAhxRAhxB(written more expansively as AcNH-RAhxRAhxB-OH in FIG. 2O) to thebackbone amines.

Example 14c: By Conjugation of Aminoalkanoic Acids to Amines of aMorpholino Oligomer Followed by Guanylation

The Morpholino Oligomer, containing free amino groups on the backboneand/or termini, was dissolved in DMSO at 75 mg/mL. Separately, anactivated Fmoc-aminoalkanoic acid solution was prepared by dissolvingthe Fmoc-protected amino acid (2 equivalents with respect to MorpholinoOligomer amines) and HBTU (1.95 equivalents with respect amines) in NMPat 100 mg/mL (with respect to amino acid). DIEA (3 equivalents withrespect to amino acid) was then added to the amino acid solution. Theactivated amino acid solution was mixed briefly and immediately added tothe Morpholino Oligomer solution. After three hours of stirring at roomtemperature, the reaction was diluted 2.33-fold with cold concentratedammonium hydroxide. The reaction was then heated at 45° C. in a sealedvessel for 18 hours, after which it was diluted 15-fold with water andpurified by SPE using an Amberchrom CG300M (Rohm and Haas; Philadelphia,Pa.) column. The SPE purification entailed loading the sample onto thecolumn at 20 mg/mL, washing the column with 4 column volumes of 1 M NaCland then 3 column volumes of water. The product was eluted by washingthe column with 3 column volumes of acetonitrile/water (1:1 v/v). Theconjugate was lyophilized and analyzed by MALDI-TOF MS and SCX HPLC. Theproduct may be guanylated and purified as per the previous examples.

The following oligomers exemplify this method:

5′-(EG3)-CTGGG+ATG+AG+ATCC+ATC+ACT-3′-(H) (SEQ ID NO: 111) was preparedusing resin with Tail. A sample (2000 OD) was converted by the methodsabove into:

5′-(EG3)-CTGGG(Ahxpip)ATG(Ahxpip)AG(Ahxpip)ATCC(G)ATC(Ahxpip)ACT-3′-(Ahx),calculated [M+H]⁺⁼7848.3 daltons. The crude product (1672 OD) recoveredafter SPE, found [M+H]⁺⁼7847.7 daltons. A portion of this material (800OD) was further converted by guanylation into5′-(EG3)-CTGGG(GuX)ATG(GuX)AG(GuX)ATCC(GuX)ATC(GuX)ACT-3′-(GuAhx),calculated [M+H]⁺⁼8100.5 daltons. The crude product recovered after SPE.found [M+H]⁺⁼8101.4 daltons, as purified by SCX chromatography to give320 OD of final product.

Example 15 Introduction of Arginine Rich Peptides into a MorpholinoOligomer

The morpholino oligomer, containing free amino groups on the backboneand/or termini, was dissolved in DMSO at 75 mg/mL. Separately, anactivated peptide solution was prepared, the peptide being 1-25 aminoacid residues in length, containing an N-terminal blocking group,preferably acetyl, and comprised of amino acids with guanidinium,hydrocarbon, or other non-nucleophilic side chains. The peptide (2equivalents with respect to morpholino oligomer amines) and HBTU (1.95equivalents with respect to amine groups) were dissolved in NMP at 100mg/mL (with respect to peptide). DIEA (2 equivalents with respect topeptide) was then added to the peptide solution. The activated peptidesolution was mixed briefly and immediately added to the morpholinooligomer solution. After three hours of stirring at room temperature,the reaction was diluted 2.33-fold with cold concentrated ammoniumhydroxide. As required, TFE was added slowly with gentle heating andmixing to redissolve the precipitated solid. The reaction was thenheated at 45° C. in a sealed vessel for 18 hours, after which it wasdiluted 15-fold with water and purified by SPE using an AmberchromCG300M (Rohm and Haas; Philadelphia, Pa.) column. The SPE purificationentailed loading the sample onto the column at 20 mg/mL, washing thecolumn with 4 column volumes of 1 M NaCl and then 3 column volumes ofwater. The product was eluted by washing the column with 3 columnvolumes of acetonitrile/water (1:1 v/v). The conjugate was lyophilizedand analyzed by MALDI-TOF MS and SCX HPLC. The product may be purifiedas per the pervious examples.

Example 16 Preparation of Morpholino Oligomers Having an Arginine RichPeptide and Backbone Guanidinium Groups

Morpholino oligomers with backbone guanidinium groups, as prepared inExample 14, were reacted with arginine rich peptides as in Example 15.The products were purified on Source 15S SCX cation exchange resin asdescribed in Example 13.

Example 17 Preparation of Morpholino Oligomers Having an Arginine RichPeptide and Backbone Amine Groups Example 17a: Protection of MorpholinoOligomer Secondary Amines as Trifluoroacetamides

41 mg of the Morpholino oligomer, an 11-mer with three backbonesecondary amines and 3′-trityl or methoxytrityl, were dissolved in 0.500mL of dimethylsulfoxide (DMSO). To the oligomer solution were added 8.2μL (5 eq) N,N-diisopropylethylamine (DIEA) followed by 44 μL (5 eq.) ofa 250 mg/mL solution of 4-nitrophenyl trifluoroacetate inN-methylpyrrolidinone (NMP). The additions of DIEA and 4-nitrophenyltrifluoroacetate were repeated four more times at 90 min intervals andthe reaction then stirred for 15 hr at room temperature. The 3′-tritylor methoxytrityl group was then removed by adding 3.76 mL (20 eq.) of a50 mM solution of 4-cyanopyridinium trifluoroacetate in2,2,2-trifluoroethanol (TFE) and stirring for 40 min. The reaction wasthen diluted to 40 mL with water and the pH adjusted to 7.5 by adding0.5 M sodium phosphate buffer, pH 7.5, dropwise. The product wasisolated by solid phase extraction using a 2 mL Amberchrom CG300Mcolumn. After loading the crude reaction mixture onto the column, thecolumn was rinsed with two column volumes of water, four column volumesof 15% acetonitrile/water (v/v), and four column volumes of 20%acetonitrile/water (v/v). The backbone-protected product with free3′-morpholine amine was then eluted with three column volumes of 1:1acetonitrile/water (v/v) and lyophilized.

Example 17b: Conjugation of Arginine Rich Peptides to MorpholinoOligomer Followed by Unmasking of Oligomer Backbone Amines

An activated peptide solution was prepared by dissolving thepeptide-acid (22.6 μmol) and HBTU (22.3 μmol) in 300 μl NMP and addingDIEA (40.8 μmol). Immediately after addition of DIEA, the peptidesolution was added to a solution of the backbone-protected Morpholinooligomer with free 3′-morpholino amine in 0.550 mL DMSO. After 180minutes at room temperature, 2 mL of concentrated ammonium hydroxidewere added to the reaction. The resulting precipitate was redissolvedwith the addition of 4 mL TFE and gentle heating and mixing. Thereaction was placed in a 45° C. oven for 15 hours. Water was then added,diluting the reaction to 40 mL. Then the solution was neutralized byadding 2 M phosphoric acid dropwise with stirring. The product wasisolated by solid phase extraction using a 2 mL Amberchrom CG300Mcolumn. After loading the crude reaction mixture onto the column, thecolumn was rinsed with four column volumes of water. The product wasthen eluted with three column volumes of 1:1 acetonitrile/water (v/v)and lyophilized.

The following oligomers exemplify this method:

5′-(EG3)-G+TGC+TCA+TGG+TGCACGG+TC-3′-(Ac(RAhxR)₄AhxB-), calculated[M+H]⁺⁼8789.3 daltons, found [M+H]⁺⁼8789.9 daltons, useful for Ebola.

5′-(EG3)-C+TTCGA+TAG+TG-3′-(trityl) was prepared using resin with Tail.A sample (994 OD) was converted by the methods above into:

5′-(EG3)-C(TFApip)TTCGA(TFApip)TAG(TFApip)TG-3′-(H), calculated[M+H]⁺⁼4368.6 daltons. The crude product recovered after SPE, found[M+H]⁺⁼4371.1 daltons. This sample was further converted by acylationwith Ac(RAhxR)₄AhxB to give5′-(EG3)-C+TTCGA+TAG+TG-3′-(Ac(RAhxR)₄AhxB-), calculated [M+H]⁺⁼6010.0daltons. The crude product (770 OD) recovered after SPE, found[M+H]⁺⁼6011.6 daltons. This was purified on SCX ion exchange at pH=6.5to provide 478 OD product, found [M+H]⁺⁼6010.7 daltons, with SCX HPLCpurity of 84.7%.

Example 18 Reductive Methylation of Morpholino Oligomer Amines

A formaldehyde solution was prepared by dissolving 0.52 gparaformaldehyde in 17 mL of 200 mM pH 8.5 sodium borate buffer withheating and stirring. The solution was heated to a gentle boil, with areflux condenser attached, for 1 hour. Heating was then ceased, thereaction mixture cooled to room temperature, and the solution continuedto stir for the duration of the methylation reaction.

A 1 M solution of sodium borohydride was prepared by cooling 10 mL of200 mM pH 8.5 sodium borate buffer on an ice bath and then dissolving0.378 g of sodium borohydride in it. The solution was kept cold on icefor the duration of the methylation reaction.

33 mg (4.6 μmol) of a 20-mer Morpholino oligomer with five secondaryamines incorporated into the backbone and a free morpholine secondaryamine at the 3′-terminus was weighed into a glass vial. The oligomer wasthen dissolved in 1 mL of 200 mM pH 8.5 sodium borate buffer and cooledto 0° C. on an ice bath with stirring. 200 μL of the formaldehydesolution (˜43 eq.) prepared above were added to the stirring Morpholinooligomer solution. Immediately after the formaldehyde addition, 40 μL ofthe 1 M sodium borohydride solution (8.7 eq.) were added. Theformaldehyde and sodium borohydride additions were repeated five timesat 30 min. intervals. After the final additions, the reaction wasstirred for 30 min. and then 4 mg of sodium borohydride added. Thereaction was then stirred for another 2 hours. Water was added to dilutethe reaction to 5 mL and the pH adjusted to 6.5 by adding 1 M phosphoricacid dropwise.

The product was isolated by solid phase extraction using a 2 mLAmberchrom CG300M column. After loading the crude reaction mixture ontothe column, the column was rinsed with four column volumes of water. Theproduct was then eluted with three column volumes of 1:1acetonitrile/water (v/v) and lyophilized.

The following oligomers exemplify this method:

5′-(EG3)-CTGGG+ATG+AG+ATCC+ATC+ACT-3′-(H) (SEQ ID NO: 111) was preparedusing resin with Tail. A sample (885 OD) was converted by the methodsabove into:

5′-(EG3)-CTGGG(Mepip)ATG(Mepip)AG(Mepip)ATCC(Mepip)ATC(Mepip)ACT-3′-(Methyl)calculated [M+H]⁺⁻7253.5 daltons. The crude product (625 OD) recoveredafter SPE. found [M+H]⁺⁼7250.5 daltons.

Example 19 Peptide Synthesis and Conjugation to PMO

Peptide synthesis of RFF (CP04073, (RFF)₃AhxβAla), SEQ ID NO:79 and RTR(CPO4074, RTRTRFLRRTAhxβAla, SEQ ID NO:80) and conjugation to PMO wereperformed using the following techniques. All the peptides of thepresent invention can be synthesized and conjugated to PMO using thesesynthetic techniques.

Peptides were synthesized by Fmoc Solid Phase Peptide Synthesis,referred to herein as SPPS. A p-benzyloxybenzyl alcohol resin was usedfor synthesis of peptides (Novabiochem, San Diego, Calif.). A typicalsynthesis cycle began with N-terminal deprotection via 20% piperidine.Then, N-α-Fmoc-protected amino acids were coupled to the growing peptidechain by activation with2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) in the presence of N,N-diisopropylethylamine (DIEA). Arginineside chains were protected with the2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) protecting groupand the t-Butyl (tBu) group for tyrosine side chains. The cycle wasrepeated until all of the amino acids were added, in a carboxy-to-aminodirection, in the desired sequence. Cleavage from the synthesis resinand side chain deprotection of the CP04073 peptide were carried outsimultaneously by treating the peptidyl-resin with a solution of 5% H₂Oand 95% trifluoroacetic acid (TFA). For the CP04074 peptide residue, acleavage cocktail of 81.5% TFA, 5% Thioanisole, 5% Phenol, 5% H₂O, 2.5%1,2-ethanedithiol (EDT) and 1% triisopropyl silane (TIS) was used forsimultaneous cleavage and side chain deprotection. Crude peptides wereisolated by precipitation using a tenfold excess of diethyl ether.Strong cation exchange HPLC utilizing Source 15S resin (AmershamBiosciences, Piscataway, N.J.) was used for purification, followed by areversed phase desalt employing Amberchrom 300M resin (Tosoh Bioscience,Montgomeryville, Pa.). Desalted peptides were lyophilized and analyzedfor identity and purity by matrix assisted laser desorption ionizationtime of flight mass spectroscopy (MALDI-TOF MS) and strong cationexchange high performance liquid chromatograph (SCX HPLC).

Attachment of the peptides at the 5′ termini of the PMO was performedvia an amide bond as follows. A C-terminally reactivepeptide-benzotriazolyl ester was prepared by dissolving the peptide-acid(15 μmol), HBTU (14.25 μmol), and HOBt (15 μmol) in 200 μL. NMP andadding DIEA (22.5 μmol). Immediately after addition of DIEA, the peptidesolution was added to 1 mL of a 12 mM solution of5′-piperazine-functionalized, 3′-acetyl-PMO in DMSO. After 180 minutesat 30° C., the reaction was diluted with a four-fold excess of water.The crude CP04074 conjugate was purified first through a CM-Sepharoseweak cation exchange column (Sigma, St. Louis, Mo.) to removeunconjugated PMO, and then through a reversed phase column (Amberchrom300M resin (Tosoh Bioscience, Montgomeryville, Pa.). The crude CP04073conjugate was purified by resolving chromatography using strong cationexchange resin (Source 30S, Amersham Biosciences, Piscataway, N.J.) toremove unconjugated PMO and excess peptide. The SCX chromatography wasfollowed by a reverse phase column (Amberchrom 300M resin (TosohBioscience, Montgomeryville, Pa.). The conjugates were lyophilized andanalyzed by MALDI-TOF MS and SCX HPLC.

Example 20 Materials and Methods for Testing Antibacterial Activity

Bacterial strains were obtained from the American Type CultureCollection (ATCC) or the E. coli Genetic Stock Center at YaleUniversity. E. coli strain AS19 was obtained from Dr. Pete Nielsen(University of Copenhagen, Denmark). All pure culture experiments weredone in 96-well plates. OD600 readings and plating of cells for CFU/mldeterminations were done in triplicate. E. coli AS19 and SM101, whichhave defects in lipopolysaccharide synthesis that result in outermembrane permeability to high MW solutes, were grown aerobically in LBbroth at 37° C., and 30° C., respectively. E. coli strain W3110 wasgrown aerobically in LB broth at 37° C.

E. coli AS19 and SM105 were grown in LB broth (supplemented with 100g/ml ampicillin for transformants that expressed luciferase) toOD₆₀₀=0.12, centrifuged (4,000×g, 10 min, 20° C.), and resuspended in 5%mucin (type II, Sigma Chemical Co., St. Louis,)/PBS to finalconcentrations as follows: AS19, 1.5×10⁸ CFU/ml; SM105, 5.7×10⁷ CFU/ml;AS19 (pT7myc-luc), 7.2×10⁹ CFU/ml. E. coli W3110 to be used for mouseinfection studies was grown in LB broth at 37° C. to OD₆₀₀=0.15,concentrated by centrifuging (4,000×g, 10 min, 20° C.) to 7×10⁹ CFU/mL,and resuspended in 5% mucin/PBS to a final concentration of 1.5×10⁸CFU/mL.

Reporter Gene.

Standard molecular biology procedures were used for all constructions.All constructs were sequenced. The acpP-luc reporter (pCNacpP-luc) wasmade by ligating a SalI-Not restriction fragment of luc with theSalI-NotI fragment of pCiNeo (Promega Corp., Madison, Wis.), removingthe adenosine from the start codon by site-directed mutagenesis, thendirectionally cloning a synthetic fragment of E. coli acpP (bp −17 to+23, inclusive, where +1 is adenosine of the start codon) between theNheI-SalI sites. Luciferase enzyme activity was measured in bacteria asdescribed (Geller, Deere at al. 2003).

Cell-Free Protein Synthesis.

Bacterial, cell-free protein synthesis reactions were performed bymixing reactants on ice according to the manufacturer's instruction(Promega Corp.). Reactions were programmed with mRNA synthesized in acell-free RNA synthesis reaction (Ambion, Inc., Austin, Tex., MEGAscriptT7 High Yield Transcription Kit) programmed with pCNacpP-luc. Whereindicated, cell-free reactions were composed with rabbit reticulocytelysate as described by the manufacturer (Promega Corp.). PMO was addedto a final concentration of either 100 nM or 200 nM as indicated. After1 hour at 37° C., the reactions were cooled on ice and luciferase wasmeasured as described (Geller, Deere et al. 2003).

Animals.

Female, 6 to 8 week old Swiss Webster or Balb/C mice (Simonsen Labs,Inc., Gilroy, Calif.) were used in all but one experiment, but identicalresults were obtained with males. Infection was established as describedin (Frimodt-Moller, Knudsen et al. 1999). Each mouse was injected IPwith 0.1 ml of bacteria resuspended in 5% mucin/PBS, then immediatelyinjected IP with 0.1 ml of PMO (3.0 mg/ml) or PBS. At various timesafter infection (as indicated in the figures), groups (n=3 to 5) of micewere injected IP with 2.0 ml PBS, and their abdomens gently massaged for2 min. Peritoneal lavage was removed and stored on ice for ˜1 hour. Thelavages were diluted in PBS and plated in triplicate on LB to determineCFU. In one experiment, blood samples (30 to 50 μL) were collected frommice via the saphenous vein, diluted in PBS, and plated in triplicate onLB to determine CFU.

Luciferase and Western Blot.

Peritoneal lavages (1.00 ml) from mice infected with AS19 (pT7myc-luc)were centrifuged (10,000×g, 2 min, 4° C.) and the supernatantsdiscarded. The pellets were resuspended in 50 μl PBS. An aliquot ofresuspended cells was mixed with an equal volume of 2×cell culture lysisreagent (Promega, Inc., Madison, Wis.) and frozen at −85° C. Frozenlysates were thawed and luciferase light production was measured induplicate in a luminometer as described (Geller, Deere et al. 2003). Asecond aliquot of the cell suspension was mixed with 2×SDS sample bufferand analyzed by western blot using 4-20% gradient Gene Mate Express Gels(ISC BioExpress, Inc., Kaysville, Utah). Blots were prepared withprimary antibody to luciferase (Cortex Biochmical, San Leandro, Calif.)or antisera to OmpA (Geller and Green 1989), secondary goat anti-rabbitIgG-horse radish peroxidase conjugate (Santa Cruz Biotechnology, Inc.,Santa, Cruz, Calif.), and ECL Western Blotting Reagent (AmershamBiosciences, Buckinghamshire, England). Film negatives were scanned anddigitized on an Kodak Image Station 440 CF. The net intensity of eachband was calculated by subtracting the mean background intensity.Luciferase protein was normalized to OmpA by dividing the net intensityof the luciferase band by the net intensity of the OmpA band in the samesample. The % inhibition was calculated by subtracting the meanluciferase/OmpA of luc PMO-treated mice from mean luciferase/OmpA ofnonsense PMO-treated mice, dividing the difference by meanluciferase/OmpA of nonsense PMO-treated mice, then multiplying by 100%.

Statistical Analysis.

Spearman's rank-order correlation was used to analyze correlationsbetween the inhibitory effects of PMO and either G+C content orsecondary structure score of each PMO. Individual mouse CFU/ml valueswere transformed logarithmically for statistical analysis using InStatstatistical software (GraphPad Software, San Diego, Calif.). Differencesin treatment group means were analysed with unpaired t test, notassuming equal variances, with Welch correction. Treatment group valueswere analysed for Gaussian distributions using the method of Kolmogorovand Smirnov, which confirmed in all analyses the normality test of thedata. A one-tailed t test was applied to differences in means betweenAcpP PMO and either PBS or scrambled PMO treatment groups, whereastwo-sided t test was applied in all other analyses.

Oligomer Sequences.

Exemplary targeting oligomers used in describing the present inventionare listed below in Table 3. The listed oligomers all target E. coli,the experimental bacterial strain used in experiments in support of theinvention. Table 4 lists the peptides of the invention and thepeptide-PMO conjugates used in experiments in support of the invention.The cationic (1-piperazino) phosphinylideneoxy linkage at each positionis indicated with a “+” in Tables 3 and 4.

TABLE 3 PMO Sequences PMO SEQ  # Sequence (5′ to 3′ ) Target ID NO 62-1TTC TTC GAT AGT GCT CAT acpP-20mer 62 62-2 TC TTC GAT AGT GCT CAT AacpP-18mer 63 62-3 C TTC GAT AGT GCT CAT acpP-16mer 64 62-4TC GAT AGT GCT CAT acpP-14mer 65 169 C TTC GAT AGT G acpP-11mer 66 C +TTCGA + TAGT + G acpP-11mer 94 379 TTC GAT AGT G acpP-10mer 67 380TTC GAT AGT acpP-9mer 68 381 TC GAT AGT acpP-8mer 69 382 TC GAT AGacpP-7mer 70 383 C GAT AG acpP-6mer 71 62-5 TTG TCC TGA ATA TCA CTTNonsense control-acpP 72 62-7 G TCC TGA ATA TCA CTTNonsense control-acpP 73 62-8 TCG TGA GTA TCA CT Nonsense control-acpP74 170 TCT CAG ATG GT Nonsense control-acpP 75 384 AAT CGG ANonsense control-acpP 76 ACG TTG AGG C Luc 77 TCC ACT TGC C luc nonsense78

TABLE 4 Peptide and Peptide-PMO Sequences Sequence Name(Amino to Carboxy Terminus, 5′ to 3′ ) SEQ ID NO RFFN-RFFRFFRFFAhxβAla-COOH 79 RTR N-RTRTRFLRRTAhxβAla-COOH 80 RFFRN-RFFRFFRFFRAhxβAla-COOH 81 KTR N-KTRTKFLKKTAhxβAla-COOH 82 KFFN-KFFKFFKFFAhxβAla-COOH 83 KFFK N-KFFKFFKFFKAhxβAla-COOH 84 (RFF)₂N-RFFRFFAhxβAla-COOH 85 (RFF)₂R N-RFFRFFRAhxβAla-COOH 86 RAhxN-RAhxAhxRAhxAhxRAhxAhxβAla-COOH 87 (RAhxR)₄N-RAhxRRAhxRRAhxRRAhxRAhxβAla-COOH 95 RFF-AcpP11N-RFFRFFRFFAhxβAla-CTTCGATAGTG-3′ 88 RTR-AcpP11N-RTRTRFLRRTAhxβAla-CTTCGATAGT G-3′ 89 RFFR-AcpP11N-RFFRFFRFFRAhxβAla-CTTCGATAGTG-3′ 90 (RFF)₂-AcpP11N-RFFRFFAhxβAla-CTTCGATAGTG 91 (RFF)₂R-AcpP11N-RFFRFFRAhxβAla-CTTCGATAGTG 92 RAhx-AcpP11 N-RAhxAhxRAhxAhxRAhxAhxβAla-93 (RAhxR)₄-AcpP11+ N-RAhxRRAhxRRAhxRRAhxRAhxβAla- 96 (RAhxR)₄N-RAhxRRAhxRRAhxRRAhxRAhxβAla-COOH 97 (RAhxR)₄-AcpP11+N-RAhxRRAhxRRAhxRRAhxRAhxβAla- 98

Example 21 Acyl Carrier Protein as an Endogenous Bacterial Gene Target

The effect of PMO was tested on an endogenous bacterial gene thatencodes acyl carrier protein, acpP, which is essential for viability(Zhang and Cronan 1996) and has been used previously to inhibitbacterial growth (Good, Awasthi et al. 2001; Geller, Deere et al. 2003).PMO from 6 to 20 bases in length and complementary to the region aroundthe start codon in mRNA for acpP (Table 3, SEQ ID NOS:62-71,respectively) were added to growing cultures of AS19 and growth at 37°C. was monitored by optical density and viable cell counts. Growthcurves were normal for all cultures except for that with the 11 basePMO, which caused significant inhibition (FIG. 3A). Slight andreproducible, but statistically insignificant inhibitions of OD occurredin cultures with the 10 and 14 base PMO. Viable cells were significantlyreduced in 8 hour cultures that contained PMO of 10, 11 or 14 bases(FIG. 3B). No reduction in CFU was apparent in cultures treated with PMOof less than 10 or more than 14 bases in length. Cultures without PMO,or with nonsense base sequences (7c, 14c and 20c; SEQ ID NOS:76, 74 and72, respectively) did not demonstrate growth inhibition.

PMO of various lengths (from 6 to 20 bases; SEQ ID NOS:62-71,respectively) and targeted to acpP were added to bacterial, cell-freeprotein synthesis reactions programmed to express an acpP-luc fusionreporter. The results (FIG. 4) show that PMO 11 to 20 bases in lengthinhibited reporter expression to about the same extent. PMO shorter than11 bases in length, or nonsense sequence controls (16c and 20c; SEQ IDNOS:73 and 72, respectively) did not inhibit luciferase expressionsignificantly.

Example 22 In Vivo Antisense Antibacterial Activity

Groups of 12 mice in each of three treatment groups were injected IPwith E. coli strain AS19, which has a genetic defect that makes itabnormally permeable to high MW solutes. Immediately followinginfection, each mouse was injected IP with 300 □g of an 11-base PMOcomplementary to acpP (PMO 169; SEQ ID NO:66), an 11-base nonsensesequence control PMO (PMO 170; SEQ ID NO:75), or PBS. Peritoneal lavageswere collected at 2, 7, 13, and 23 hours post-infection, and plated forbacteria. The results show that at all times analyzed, the acpPPMO-treated mice had significantly (P<0.05) lower CFU than the micetreated with either nonsense PMO or PBS (FIG. 5). The differencesbetween the acpP PMO-treated group and the nonsense PMO control rangesfrom 39-fold at 2 hours to 600-fold at 23 hours.

The same PMOs were again tested, except with E. coli strain SM105, whichhas a normal outer membrane. AcpP PMO reduced CFU by 84% compared tononsense PMO at 12 hours post-infection. There was no reduction of CFUat 2, 6, or 24 hours (FIG. 6). Mice were injected with a second dose at24 hours post-infection. By 4 hours post-infection the CFU of acpPPMO-treated mice were 70% lower than the CFU of nonsense PMO-treatedmice (FIG. 6).

The above results with acpP and nonsense PMOs suggest that inhibitionwas sequence specific. To demonstrate directly a sequence-specificeffect, mice were infected with an E. coli AS19 that expresses fireflyluciferase, then treated at 0 and 13 hours post-infection with a PMO(luc; SEQ ID NO:77) complementary to the region around the start codonof the luciferase transcript, or a nonsense PMO (luc nonsense; SEQ IDNO:78). Peritoneal lavages were collected at 13 and 22 hourspost-infection and analyzed for CFU, luciferase activity, and luciferaseand OmpA protein by western immuno-blot analysis. As expected, theresults show no inhibition of growth with the luc PMO treatment comparedto nonsense PMO treatment (Table 5). Luciferase activity in samples fromluc PMO-treated mice was inhibited 53% and 46% at 13 and 22 hours,respectively, compared to samples from nonsense PMO-treated mice (Table5).

Western blot analysis agreed closely with the results of luciferaseactivity. In samples from luc PMO-treated mice, there was a 68% and 47%reduction in the amount of luciferase protein at 13 and 22 hours,respectively, compared to samples from nonsense PMO-treated mice (Table5).

TABLE 5 Gene Specific Inhibition Luciferase Activity Western Blot TimeRLU/CFU Luc/OmpA after Mean Mean PMO treatment CFU/ml (SEM) % (SEM) %Treatment (h) (×10⁶) n = 8 P Inhibition n = 7-8 P Inhibition Luc 13 6.32.90 .0035 53 0.122 .0002 68 (0.629) (.0312) Nonsense 13 4.3 6.19 00.382 0 (0.773) (.0296) Luc 22 0.96 3.20 .0093 46 0.147 .0145 57 (0.582)(.0363) Nonsense 22 0.39 8.12* 0 0.339 0 (1.94) (.0668)

Example 23 Enhanced Anti-Bacterial Properties of Peptide-Conjugated PMO

PMO conjugated at the 5′ terminus with a series of three differentpeptides were evaluated for their antibacterial properties. The 11 PMOthat targets the E. coli acpP gene (SEQ ID NO:66) was used as theantisense oligomer moiety and conjugated to either the RFF, RTR or RAhxpeptides (SEQ ID NOS:79-80 and SEQ ID NO:87, respectively) to producepeptide-conjugated PMOs (P-PMOs) RFF-AcpP11, RTR-AcpP11 and RAhx-AcpP11(SEQ ID NOS:88-89 and SEQ ID NO:93, respectively).

Four laboratory strains of E. coli (W3110, MIC2067, DH5a, SM105) weretreated with 20 μM RFF-AcpP11 PPMO (SEQ ID NO:88), 20 μM free RFFpeptide (SEQ ID NO:79) with unconjugated AcpP11 μMO (SEQ ID NO:66), orreceived no treatment (control) in LB broth, then were incubated at 37°C. for 24 hours. Every hour for 8 hours then at 24 hours the OD600values (turbidity measurement) of the cultures were measured using aspectrophotometer. After 8 hours, aliquots of the cultures were dilutedthen plated onto LB agar plates and incubated for 24 hours at 37° C.After incubation, the colonies were counted by hand and the colonyformation units/mL (CFU/mL) for each treatment were calculated. FIGS. 7to 10 show the 24 hour growth curves for strains W3110, MIC2067, DH5αand SM105, respectively, in the presence of the RFF-AcpP11 P-PMO. FIG.11 shows the CFU/ml after 8 hours treatment for the four strains of E.coli.

Using identical conditions as described above, E. coli/W3110 was treatedwith three different P-PMOs (RAhx-AcpP11, RTR-AcpP11 and RFF-AcpP11) orreceived no treatment. FIG. 12 shows the CFU/ml after 8 hours oftreatment with the three different P-PMOs compared to no treatment.

E. coli W3110 was treated with a short dilution series of RFF peptide at5 μM, 20 μM, 50 μM and 100 μM, 20 μM RFF free peptide mixed with AcpP11μMO, 20 M RFF-AcpP11 P-PMO, 10 μM ampicillin or no treatment. FIG. 13shows the CFU/ml after 8 hours for each treatment. This data stronglysupports the conclusion that only the P-PMO and ampicillin showedantibacterial activity and that the delivery peptide must be conjugatedto the PMO for this effect. Furthermore, the RFF-AcpP11 P-PMOdemonstrates an approximately 10 fold improved antibacterial activity at10 μM compared to ampicillin at the same concentration.

A series of dose-response experiments were performed where E. coli W3110was exposed to RFF-AcpP11, RTR-AcpP11, and ampicillin in pure culture.E. coli W3110 was treated with a dilution series of RFF-AcpP11,RTR-AcpP11 or ampicillin (80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM) orreceived no treatment. Determination of 50% inhibitory concentrationvalues (IC₅₀) for RFF-AcpP11, RTR-AcpP11, and ampicillin were made usingstandard methods. FIG. 14 shows the dose response curves for each of thetwo PPMOs compared to ampicillin and the associated IC₅₀ values of 3.7,12.1 and 7.7 mM for RFF-AcpP11, RTR-AcpP11 and ampicillin, respectively.

The sensitivity of Salmonella typhimurium 1535 and a clinically isolatedenteropathogenic strain of E. coli (EPEC strain 0127:H6) to RFF-AcpP11P-PMO in culture was determined. The target sequences for AcpP11 (SEQ IDNOS:2 and 8) in S. typhimurium and E. coli are identical. Both strainswere treated with 2□M of RFF-AcpP11 P-PMO, RFF free peptide mixed withunconjugated AcpP11 μMO, AcpP11scr PMO, RFF-Scr P-PMO, RFF free peptidemixed with unconjugate AcpP11 μMO or no treatment. FIGS. 15 and 16 showthe CFU/mL after 8 hours treatment for S. typhimurium and EPEC strain0127:H6. This data clearly demonstrate the utility of the RFF-AcpP11compound as an antibacterial agent against clinically relevant bacterialisolates.

Example 24 Antibacterial Activity of Peptide-Conjugated PMO TargetingBurkholderia and Pseudomonas Species

Burkholderia cenocepacia growth in the presence of peptide-conjugatedPMO was determined using the compounds of the invention. A stationaryphase culture of B. cenocepacia was diluted to 5×10⁵ cfu/ml inMueller-Hinton broth. The peptide-conjugated PMO that target the acpPand gyrA genes were added to identical cultures to a final concentrationof 200 μmol/L. The cultures were grown aerobically at 37° C. and opticaldensity was monitored. After 36 hours, each culture was diluted andplated to determine viable cell count as colony forming units per ml(CFU/ml). All peptide-conjugated PMOs had the same peptide attached tothe 5′ end, which was (RFF)₃RAhxβAla(SEQ ID NO:81). All PMOs were 11bases in length and targeted to regions around the start codon of acylcarrier protein (acpP) or the DNA gyrase subunit A (gyrA) as describedabove. Scr is a negative control, scrambled base sequencepeptide-conjugated PMO that has no complementary target in B.cenocepacia. FIG. 17 shows the inhibition of Burkholderia cenocepaciagrowth, as measured by optical density, by the gyrA and acpPpeptide-conjugated PMO compared to the Scr control. The viable cellcount after 36 hours treatment was less than 1×10⁴ CFU/ml for thepeptide-conjugated PMOs targeting the acpP and gyrA genes whereas forthe Scr control peptide-conjugated PMO the viable cell count was 8.5×10⁷CFU/ml. Similar results were obtained using these PMO against B.multivorans and B. gen.II.

Similar experiments targeting Pseudomonas aeruginosa using RFFR (SEQ IDNO:81) conjugated to a PMO that targets the P. aeruginosa acpP gene. TheRFFR-AcpP PMO targeted to acpP was added to a growing culture of P.aeruginosa in Mueller-Hinton broth at a 20 micromolar concentration.Aerobic growth at 37° C. was measured by optical density at 600 nm. Theresults shown in FIG. 18 show complete inhibition of growth and thescrambled base sequence control had no effect on growth.

Example 25 Enhanced In Vivo Anti-Bacterial Properties of aPeptide-Conjugated PMO Containing Cationic Intersubunit Linkages(P-PMO+)

A PMO having cationic intersubunit linkages and conjugated with anarginine rich peptide was evaluated for its antibacterial properties ina dose-response study. The 11mer cationic PMO+ that targets the E. coliacpP gene (SEQ ID NO: 94) was used as the antisense oligomer moiety andconjugated at the 3′ terminus to the (RAhxR)₄ peptide (SEQ ID NO: 95) toproduce the cationic peptide-conjugated (RAhxR)₄-AcpP11+P-PMO+(SEQ IDNO: 96).

Groups of 2 to 4 mice in each of six treatment groups were injected IPwith 1.5×10⁷ CFU of E. coli strain W3110. Each mouse was then injectedIP with water control or a 1, 10, 30, 100, or 300 μg dose of(RAhxR)₄-AcpP11+ administered both at 15 minutes and 12 hours postinfection. Blood samples were collected and plated to determine CFU/mLat 2, 6, 12, 24, and 48 hours post infection. Body temperature wasrecorded as an objective criterion of terminal illness at the same timepoints as blood collection, with a threshold body temperature of 27.9°C. or below predicting terminal illness and ensuing death. Mousesurvival was also tracked at same the time points as blood collectionand scored as a death if body temperature dropped below 28° C. or ifmouse was found dead.

The results show minor efficacy in the 1 g group while full protectionup to termination of the study at 48 hours post infection was observedin all groups of mice administered 10 g and above (FIG. 19A). Adose-dependent reduction in CFU was observed, ranging from 0.5 to 4orders of magnitude below water control for the 1 g and 300 g groups,respectively, by 2 hours post infection. By 12 hours post infection, CFUwas reduced from 2 to 4 orders of magnitude below water control for the10 g and 300 g groups, respectively (FIG. 19B). At 48 hours postinfection, body temperatures ranged from 36.3 to 37.4° C. for the 10 gand 300 g groups, respectively, while all mice in the water control and1 g groups were dead by 12 and 24 hours post infection, respectively(FIG. 19C). No treatment-related toxicity was observed in any of thegroups.

Although the application has been described with respect to particularembodiments, methods, and applications, it will be appreciated thatvarious changes and modifications may be made without departing from theinvention.

SEQUENCE LISTING

Sequence  SEQ Name (5′ to 3′) or (amino to carboxyl) ID NO E. coli ftsZGAGAGAAACTATGTTTGAACCAATGGAACTT  1 E. coli acpPATTTAAGAGTATGAGCACTATCGAAGAACGC  2 E. coli gyrATAGCGGTTAGATGAGCGACCTTGCGAGAGAA  3 E. coli O157:H7 ftsZGAGAGAAACTATGTTTGAACCAATGGAACTT  4 E. coli O157:H7 acpPATTTAAGAGTATGAGCACTATCGAAGAACGC  5 E. coli O157:H7 gyrATAGCGGTTAGATGAGCGACCTTGCGAGAGAA  6 S. typhimurium ftsZGAGAGAGATTATGTTTGAACCTATGGAACTA  7 S. typhimurium acpPATTTAAGAGTATGAGCACTATCGAAGAACGC  8 S. typhimurium gyrATAGCGGTTAGATGAGCGACCTTGCGAGAGAA  9 P. aeruginosa ftsZGAGAGGGGAAATGTTTGAACTGGTCGATAAC 10 P. aeruginosa acpPAAAACAAGGTATGAGCACCATCGAAGAACGC 11 P. aeruginosa gyrACAGGCTTCTCATGGGCGAACTGGCCAAAGAA 12 V. cholera ftsZGAGATAACACATGTTTGAACCGATGATGGAA 13 V. cholera acpPACTATATTGGATGGTTTATATGTCTATCTCT 14 V. cholera gyrATAATGGCTCTATGAGCGATCTAGCTAAAGAG 15 N. ghonorrhoea ftsZGAGTTTTTGAATGGAATTTGTTTACGACGT 16 N. ghonorrhoea acpPAACGACTGATATGTCAAACATCGAACAACA 17 N. ghonorrhoea gyrACATTGAAACCATGACCGACGCAACCATCCG 18 S. aureus ftsZGGAAATTTAAATGTTAGAATTTGAACAAGGA 19 S. aureus gyrAGGAACTCTTGATGGCTGAATTACCTCAATCA 20 S. aureus fmhBATCATAAATCATGGAAAAGATGCATATCAC 21 M. tuberculosis ftsZCTCTAAGCCTATGGTTGAGGTTGAGAGTTTG 22 M. tuberculosis acpPCCCGGGCGCGATGTGGCGATATCCACTAAGT 23 M. tuberculosis gyrACGAGGAATAGATGACAGACACGACGTTGCCG 24 M. tuberculosis pimAGGAAAGCCTGATGCGGATCGGCATGATTTG 25 M. tuberculosis cysS2CTGGCACGTCGTGACCGATCGGGCTCGCTT 26 H. pylori ftsZGAATGTGGCTATGGTTCATCAATCAGAGATG 27 H. pylori acpPAGTTTTAATTATGGCTTTATTTGAAGATATT 28 H. pylori gyrAAGGGAGACACATGCAAGATAATTCAGTCAAT 29 S. pneumoniae ftsZAAAATAAATTATGACATTTTCATTTGATACA 30 S. pneumoniae acpPGAGTCCTATCATGGCAGTATTTGAAAAAGTA 31 S. pneumoniae gyrAGCATTTATTAATGCAGGATAAAAATTTAGTG 32 T. palladium ftsZTGGGAGGGGAATGATGAATATAGAGCTTGCA 33 T. palladium acpPTGCCCCGTGGATGAGTTGTTCTTAAGAATGA 34 T. palladium gyrATGCCCGCCCTATGGAAGAAATTAGCACCCCA 35 C. trachomatis acpPGGATCATAGGATGAGTTTAGAAGATGATGTA 36 C. trachomatis gyrAAAACGAACTTATGAGCGACCTCTCGGACCTA 37 B. henselae ftsZAGGCAAATTAATTGGTAAAAAATTAGAGAG 38 B. henselae acpPGGATTTCAACATGAGTGATACAGTAGAGCG 39 B. henselae gyrAGTCTAAAGCTGTGACAGATCTAAACCCGCA 40 H. influenza ftsZGAGAACATCAATGCTATACCCAGAGTACCCT 41 H. influenza acpPGGAAAAACAAATGAGTATTGAAGAACGCGTG 42 H. influenza gyrAAGGAATACCAATGACGGATTCAATCCAATCA 43 L. monocytogenes ftsZAGGCAATAATATGTTAGAATTTGACACTAG 44 L. monocytogenes acpPCGAACGCATAAAACTTTATGTGACCGGATA 45 L. monocytogenes gyrATTCTCTAACAATGGCAGAAACACCAAATCA 46 Y. pestis ftsZGAGAGAAACTATGTTTGAACCTATGGAACT 47 Y. pestis acpPATTTAAGAGTATGAGCACTATCGAAGAACG 48 Y. pestis gyrATAGCGGCTCAATGAGCGACCTTGCCAGAGA 49 B. anthracis ftsZGGATTTCGACATGTTAGAGTTTGATACTAC 50 B. anthracis acpPGGTGAATGGAATGGCAGATGTTTTAGAGCG 51 B. anthracis gyrAGTGCTCGTTGATGTCAGACAATCAACAACA 52 B. mallei ftsZGGAGGCAACAATGGAATTCGAAATGCTGGA 53 B. mallei acpPCGGAGGGGTAATGGACAACATCGAACAACG 54 B. mallei gyrAATACGGATACATGGATCAATTCGCCAAAGA 55 B. pseudomallei ftsZGGAGGCAACAATGGAATTCGAAATGCTGGA 56 B. pseudomallei acpPCGGAGGGGTAATGGACAACATCGAACAACG 57 B. pseudomallei gyrAATACGGATACATGGATCAATTCGCCAAAGA 58 F. tularensis ftsZGGAGTAAAATATGTTTGATTTTAACGATTC 59 F. tularensis acpPAGGAAAAAATATGAGTACACATAACGAAGA 60 F. tularensis gyrAGCGATAACTAATGTCTATAATTACTAAAGA 61 62-1 TTCTTCGATAGTGCTCATAC 62 62-2TCTTCGATAGTGCTCATA 63 62-3 CTTCGATAGTGCTCAT 64 62-4 TCGATAGTGCTCAT 65169 (acpP , +5 to +15) CTTCGATAGTG 66 (acpP+, +5 to +15) C + TTCGA +TAGT + G 379 TTCGATAGTG 67 380 TTCGATAGT 68 381 TCGATAGT 69 382 TCGATAG70 383 CGATAG 71 62-5 TTGTCCTGAATATCACTTCG 72 62-7 GTCCTGAATATCACTT 7362-8 TCGTGAGTATCACT 74 170 TCTCAGATGGT 75 384 AATCGGA 76 Luc ACGTTGAGGC77 Luc-nonsense TCCACTTGCC 78 RFF N-RFFRFFRFFAhxβAla-COOH 79 RTRN-RTRTRFLRRTAhxβAla-COOH 80 RFFR N-RFFRFFRFFRAhxβAla-COOH 81 KTRN-KTRTKFLKKTAhxβAla-COOH 82 KFF N-KFFKFFKFFAhxβAla-COOH 83 KFFKN-KFFKFFKFFKAhxβAla-COOH 84 (RFF)₂ N-RFFRFFAhxβAla-COOH 85 (RFF)₂RN-RFFRFFRAhxβAla-COOH 86 RAhx N-RAhxAhxRAhxAhxRAhxAhxβAla-COOH 87RFF-AcpP11 N-RFFRFFRFFAhxβAla-CTTCGATAGTG 88 RTR-AcpP11N-RTRTRFLRRTAhxβAla-CTTCGATAGTG 89 RFFR-AcpP11N-RFFRFFRFFRAhxβAla-CTTCGATAGTG 90 (RFF)₂-AcpP11N-RFFRFFAhxβAla-CTTCGATAGTG 91 (RFF)₂R-AcpP1N-RFFRFFRAhxβAla-CTTCGATAGTG 92 RAhx-AcpP11 N-RAhxAhxRAhxAhxRAhxAhxβAla-93 acpP (-4 to +7) TGCTCATACTC 94 acpP (+1 to +11) ATAGTGCTCAT 95acpP (+11 to +21) GCGTTCTTCCG 96 (RAhxR)₄N-RAhxRRAhxRRAhxRRAhxRAhxβAla-COOH 97 (RAhxR)₄-AcpP11+N-RAhxRRAhxRRAhxRRAhxRAhxβAla- 98

1. A method for enhancing the antibacterial activity of an antisense oligonucleotide composed of morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, where the oligonucleotide contains between 10-20 bases and a targeting sequence of at least 10 contiguous bases complementary to a bacterial RNA target, and where binding of the oligonucleotide to the RNA target region is effective to inhibit growth of an infectious bacterium in a mammalian host, comprising one or both of steps (a) and (b): (a) conjugating to the oligonucleotide, a carrier peptide (i) containing 8-14 amino acids composed of the subsequences selected from the group represented by XXY, XY, XZZ and XZ, and permutations of the subsequences, where: each X subunit independently represents arginine or an arginine analog, said analog being a cationic α-amino acid comprising a side chain of the structure R¹N═C(NH₂)R², where R¹ is H or R′; R² is R′, NH₂, NHR′, or NR′₂, where R′ is lower alkyl or lower alkenyl and may further include oxygen or nitrogen; R¹ and R² may together form a ring; and the side chain is linked to said amino acid via R¹ or R², each Y subunit independently represents a neutral linear amino acid —C(O)—(CHR″)_(m)—NH—, where (i) m is 1 to 7 and each R″ is independently H or methyl, and Z is an α-amino acid having a neutral side chain selected from a substituted or unsubstituted aralkyl, and (ii) coupled to the oligonucleotide at the peptide's C terminus; and (b) modifying the oligonucleotide to contain 20%-50% intersubunit cationic linkages that are positively charged at physiological pH.
 2. The method of claim 1, wherein the carrier peptide is represented by the sequence (RY′R)_(n) and (RY′)_(n), where R is arginine and Y′ is a linear alkanoic acid having 2-7 carbon atoms in its backbone chain.
 3. The method of claim 1, wherein the carrier peptide is linked at its C-terminus to the 5′ end of the oligonucleotide through a one- or two-amino acid linker.
 4. The method of claim 3, wherein the linker is AhxβAla, where Ahx is 6-aminohexanoic acid and βAla is β-alanine.
 5. The method of claim 1, wherein the peptide is represented by (RAhxR)₄,where R is arginine and Ahx is 6-amino hexanoic acid.
 6. The method of claim 1, wherein the peptide is represented by (RAhx)₆, where R is arginine and Ahx is 6-amino hexanoic acid.
 7. The method of claim 1, wherein said oligonucleotide has approximately equal-length 5′, 3′ and center regions, and the percentage of cationic linkages in said center region is greater than about 70%.
 8. The method of claim 1, wherein step (a) of the method is effective to enhance the anti-bacterial activity of an uncharged oligonucleotide, as measured by inhibition in bacterial growth in vitro over an eight-hour period, by a factor of at least 10 relative to the measured inhibition of the uncharged oligonucleotide in the absence of the carrier peptide.
 9. The method of claim 8, wherein step (a) and step (b) of the method are effective to enhance the anti-bacterial activity of an uncharged oligonucleotide, as measured by inhibition in bacterial growth in vitro over an eight-hour period, by a factor of at least 10 relative to the measured inhibition of the uncharged oligonucleotide in the absence of the carrier peptide.
 10. The method of claim 1, morpholino subunits in the oligonucleotide are joined by phosphorodiamidate linkages, in accordance with the structure:

where Z is S or O, X is NR¹¹R¹² or OR¹⁶, Y is O or NR¹⁷, and each said linkage is selected from: (a) uncharged linkage (a), where each of R¹¹, R¹², R¹⁶ and R¹⁷ is independently selected from hydrogen and lower alkyl; (b1) cationic linkage (b1), where X is NR¹¹R¹² and Y is O, and NR¹¹R¹² represents an optionally substituted piperazino group, such that R¹¹R¹² is —CHR^(a)CHR^(a)N(R¹³)(R¹⁴)CHR^(a)CHR^(a), where each R^(a) is independently H or CH₃, R¹⁴ is H, CH₃ or an electron pair, and R¹³ is selected from H, lower alkyl, C(═NH)NH₂, Z-L-NHC(═NH)NH₂, and [C(O)CHR′″NH]_(m)H, where Z is carbonyl (C(O)) or a direct bond, L is an optional linker up to 18 atoms in length having bonds selected from alkyl, alkoxy, and alkylamino, R′″ is a side chain of a naturally occurring amino acid or a one- or two-carbon homolog thereof, and m is 1 to 6; (b2) cationic linkage (b2), where X is NR¹¹R¹² and Y is O, R¹¹ is H or CH₃, and R¹² is LNR¹³R¹⁴R¹⁵, where L, R¹³, and R¹⁴ are as defined above, and R¹⁵ is H, lower alkyl, or lower (alkoxy)alkyl; and (b3) cationic linkage (b3), where Y is NR¹⁷ and X is OR¹⁶, and R¹⁷ is LNR¹³R¹⁴R¹⁵, where L, R¹³, R¹⁴ and R¹⁵ are as defined above, and R¹⁶ is H or lower alkyl; and at least one said linkage is selected from cationic linkages (b1), (b2), and (b3).
 11. The method of claim 10, wherein each of R¹¹ and R¹², in linkages of type (a), is methyl.
 12. The method of claim 10, wherein at least one linkage is of type (b1), where each R^(a) is H, R¹⁴ is H, CH₃, or an electron pair, and R¹³ is selected from H, CH₃, C(═NH)NH₂, and C(O)-L-NHC(═NH)NH₂.
 13. The method of claim 10, wherein at least one linkage is of type (b1), where each R^(a) is H, R¹⁴ is an electron pair, and R¹³ is selected from C(═NH)NH₂ and C(O)-L-NHC(═NH)NH₂.
 14. (canceled)
 15. The method of claim 13, wherein R¹³ is C(O)-L-NHC(═NH)NH₂, and L is a hydrocarbon having the structure —(CH₂)_(n)—, where n is 1 to
 12. 16. The method of claim 1, wherein at least one linkage is of type (b1), where each R^(a) is H, and each of R¹³ and R¹⁴ is independently H or CH₃.
 17. The method of claim 1, for treating a gram-negative bacterial infection, wherein the targeting sequence is complementary to a target sequence containing or within 20 bases, in a downstream direction, of the translational start codon of a bacterial mRNA that encodes acyl carrier protein (acpP).
 18. The method of claim 1, for treating a gram-negative bacterial infection, wherein the targeting sequence is complementary to a target sequence containing or within 20 bases, in a downstream direction, of the translational start codon of a bacterial mRNA that encodes gyrase A subunit (gyrA).
 19. In an antisense oligonucleotide useful for treating a bacterial infection and composed of morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, where the oligonucleotide contains between 10-20 bases and a targeting sequence of at least 10 contiguous bases complementary to a bacterial RNA target, and where binding of the oligonucleotide to the RNA target region is effective to inhibit growth of the infectious bacterium, an improvement that enhances the antibacterial active of the oligonucleotide at least 10-fold, comprising one or both of (a) and (b); (a) conjugated to the oligonucleotide, a carrier peptide (i) containing 8-14 amino acids composed of the subsequences selected from the group represented by XXY, XY, XZZ and XZ, and permutations of the subsequences, where: each X subunit independently represents arginine or an arginine analog, said analog being a cationic α-amino acid comprising a side chain of the structure R¹N═C(NH₂)R², where R¹ is H or R′; R² is R′, NH₂, NHR′, or NR′₂, where R′ is lower alkyl or lower alkenyl and may further include oxygen or nitrogen; R¹ and R² may together form a ring; and the side chain is linked to said amino acid via R¹ or R², each Y subunit independently represents a neutral linear amino acid —C(O)—(CHR″)_(m)—NH—, where (i) m is 1 to 7 and each R″ is independently H or methyl, and Z is an α-amino acid having a neutral side chain selected from a substituted or unsubstituted aralkyl, and (ii) coupled to the oligonucleotide at the peptide's C terminus; and (b) the presence in the oligonucleotide of 20%-50% intersubunit cationic linkages that are positively charged at physiological pH.
 20. The improvement of claim 19, wherein the carrier peptide is represented by the sequence (RY′R)_(n) and (RY′)_(n), where R is arginine and Y′ is a linear alkanoic acid having 2-7 carbon atoms in its backbone chain. 